Cell regulatory genes, encoded products, and uses related thereto

ABSTRACT

This application describes the cloning of p63, a gene at chromosome 3q27-29, that bears homology to the tumor suppressor p53. The p63 gene encodes at least six different isotypes. p63 was detected in a variety of human and mouse tissue and demonstrates remarkably divergent activities, such as the ability to transactivate p53 reporter genes and induce apoptosis. Isotypes of p63 lacking a transactivation domain act as dominant negatives towards the transactivation by p53 and p63.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/174,493, filed Oct. 15, 1998, which claims the benefit of U.S.Provisional Application Nos. 60/087,216 filed May 29, 1998, and60/062,076 filed Oct. 15, 1997. Each of these applications is herebyincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Support for research leading to subject matter disclosed in thisapplication was provided in part by the National Institutes of HealthGrant No. 5R01GM052027. Accordingly, the United States Government hascertain rights with respect to subject matter of this application.

BACKGROUND

Neoplasia is characterized by deregulated cell growth and division.Inevitably, molecular pathways controlling cell growth must interactwith those regulating cell division. It was not until very recently,however, that experimental evidence became available to bring suchconnection to light. Cyclin A was found in association with theadenovirus oncoprotein E1A in virally transformed cells (Giordona et al.Cell 58:981 (1989); and Pines et al. Nature 346:760 (1990)). Thecell-cycle gene implicated most strongly in oncogenesis thus far is thehuman cyclin D1. It was originally isolated through geneticcomplementation of yeast G₁ cyclin deficient strains (Xiong et al. Cell65:691 (1991); and Lew et al. Cell 66:1197 (1991)), as cellular geneswhose transcription is stimulated by CSF-1 in murine macrophages(Matsushine et al. Cell 65:701 (1991)) and in the putative oncogenePRAD1 rearranged in parathyroid tumors (Montokura et al. Nature 350:512(1991).

However, the creation of a mutant oncogene is only one of therequirements needed for tumor formation; tumorigenesis appears to alsorequire the additional inactivation of a second class of critical genes:the “anti-oncogenes” or “tumor-suppressing genes.” Tumor suppressorgenes are a family of genes that negatively regulate cell growth and arelost or inactivated in most cancers. In their natural state these genesact to suppress cell proliferation. Damage to such genes leads to a lossof this suppression, and thereby results in tumorigenesis. Thus, thederegulation of cell growth may be mediated by either the activation ofoncogenes or the inactivation of tumor-suppressing genes (Weinberg, R.A., (September 1988) Scientific Amer. pp 44-51).

Oncogenes and tumor-suppressing genes have a basic distinguishingfeature. The oncogenes identified thus far have arisen only in somaticcells, and thus have been incapable of transmitting their effects to thegerm line of the host animal. In contrast, mutations intumor-suppressing genes can be identified in germ line cells, and arethus transmissible to an animal's progeny.

The classic example of a hereditary cancer is retinoblastomas inchildren. The incidence of the retinoblastomas is determined by a tumorsuppressor gene, the retinoblastoma (RB) gene (Weinberg, R. A.,(September 1988) Scientific Amer. pp 44-51; Hansen et al. (1988) TrendsGenet. 4:125-128). Individuals born with a lesion in one of the RBalleles are predisposed to early childhood development ofretinoblastomas. Inactivation or mutation of the second RB allele in oneof the somatic cells of these susceptible individuals appears to be themolecular event that leads to tumor formation (Caveneee et al. (1983)Nature 305:799-784; Friend et al. (1987) PNAS 84:9059-9063).

The RB tumor-suppressing gene has been localized onto human chromosome13. The mutation may be readily transmitted through the germ line ofafflicted individuals (Cavenee, et al. (1986) New Engl. J. Med314:1201-1207). Individuals who have mutations in only one of the twonaturally present alleles of this tumor-suppressing gene are predisposedto retinoblastoma. Inactivation of the second of the two alleles is,however, required for tumorigenesis (Knudson (1971) PNAS 68:820-823).

A second tumor-suppressing gene is the p53 gene (Green (1989) Cell56:1-3; Mowat et al (1985 Nature 314:633-636). The protein encoded bythe p53 gene is a nuclear protein that forms a stable complex with boththe SV40 large T antigen and the adenovirus E1B 55 kd protein. The p53gene product may be inactivated by binding to these proteins.

Based on cause and effect analysis of p53 mutants, the functional roleof p53 as a “cell-cycle checkpoint”, particularly with respect tocontrolling progression of a cell from G1 phase into S phase, hasimplicated p53 as able to directly or indirectly affect cell cyclemachinery. The first firm evidence for a specific biochemical linkbetween p53 and the cell-cycle comes a finding that p53 apparentlyregulates expression of a second protein, p21, which inhibitscyclin-dependent kinases (CDKs) needed to drive cells through thecell-cycle, e.g. from G1 into S phase. For example, it has beendemonstrated that non-viral transformation, such as resulting at leastin part from a mutation of deletion of the p53 tumor suppressor, canresult in loss of p21 from cyclin/CDK complexes. As described by Xionget al. (1993) Nature 366:701-704, induction of p21 in response to p53represents a plausible mechanism for effecting cell-cycle arrest inresponse to DNA damage, and loss of p53 may deregulate growth by loss ofthe p21 cell-cycle inhibitor.

More recently, researchers discovered yet another tumor suppressinggene, p73, which closely resembles p53. Not only does this protein beara strong structural identity with p53, it also possess similarfunctional attributes. For instance, this protein disclosed thegrowth-inhibiting and apoptosis promoting effects, it triggered p21production, suggesting thereby that it inhibited cell growth through thesame pathway as that used by p53. Here, we describe the discovery of anovel family of cell regulatory genes, the p-63 family, which exhibitsconsiderable sequence identity with p53 and p73, and appears to possesssimilar functional attributes.

SUMMARY

The p53 tumor suppressor protein is involved in multiple centralcellular processes, including transcription, DNA repair, genomicstability, senescence, cell cycle control and apoptosis. p53 isfunctionally inactivated by structural mutations, interaction with viralproducts, and endogenous cellular mechanisms in the majority of humancancers. In fact, the p53 protein is one of the most frequently mutatedtumor suppressor to be identified in human cancers. More than 50% ofprimary human tumor cells over-express a variety of mutant p53 forms.p73 which shares considerable structural identity maps to chromosomalregion 1p36, a region which is frequently deleted in neuroblastoma andother tumors.

Here we describe a third family of cell regulatory genes, encoding thep63-family of proteins which also demonstrate considerable structural orsequence identity to the DNA-binding, oligomerization, andtransactivation domains of p53. p63 differs from p53 in that multiplep63 transcripts yielding six major protein products have been discoveredby cDNA cloning. For example, the six major p63 products are listed inFIG. 2B. It was found that unlike p53, the p63 gene encodes multipleisotypes with remarkably divergent abilities. For instance, p63 variantspossessing the N-terminus, i.e., TAp63γ, showed strong transactivationand cell-death inducing abilities. TAp63γ transactivates p53 reportergenes and may induce apoptosis. In addition, it was found that thepredominant p63 isotypes in many epithelial tissues lack an acidicN-terminus corresponding to the transactivation domain of p53. p63variants which lack the transactivation domain, i.e., ΔNp63α, ΔNp63γ,suppressed transactivation by both p53 and p63. Additionally, thesevariants lacking the N-terminus may possibly regulate growth and mayplay an essential role in the regenerative processes, particularlyregeneration of epithelial tissue. In one aspect, the inventiondiscloses that these truncated p63 variants can act as dominant-negativeagents towards transactivation by p53, thereby suggesting thepossibility of physiological interactions amongst members of the p53family. Examples of these variants include the disclosed ΔNp63α andΔNp63γ. Thus, in one embodiment, the p63 family of cell-regulatoryproteins are involved in the modulation of cell growth or regulate thegrowth phenotype of a cell.

One aspect of the invention features a substantially pure preparation ofa cell regulatory protein, or a fragment thereof, the full-length formof the cell regulatory protein having an amino acid sequence at least70% homologous to the amino acid sequence represented in one of SEQ IDNos. 13-24; the polypeptide has an amino acid sequence at least 80%homologous to the amino acid sequence represented in one of SEQ ID Nos.13-24; the polypeptide has an amino acid sequence at least 90%homologous to the amino acid sequence represented in one of SEQ ID Nos.13-24; the polypeptide has an amino acid sequence at least 95%homologous to the amino acid sequence represented in one of SEQ ID Nos.13-24; the polypeptide has an amino acid sequence identical to the aminoacid sequence represented in one of SEQ ID Nos. 13-24. In a preferredembodiment: the fragment comprises at least 5 contiguous amino acidresidues of SEQ ID Nos. 13-24; the fragment comprises at least 20contiguous amino acid residues of SEQ ID Nos. 13-24; the fragmentcomprises at least 50 contiguous amino acid residues of SEQ ID Nos.13-24.

In yet another embodiment, the fragment includes the DNA binding domainof p63 and comprises at least 5 contiguous amino acid residues of SEQ IDNos. 13-24; the fragment includes the DNA binding domain of p63 andcomprises at least 20 contiguous amino acid residues of SEQ ID Nos.13-24; the fragment includes the DNA binding domain of p63 and comprisesat least 50 contiguous amino acid residues of SEQ ID Nos. 13-24.

The DNA binding domain of these nucleic acid transcripts are remarkablyconserved. The six variants demonstrate 100% identity in this region andthis domain also demonstrates considerable identity to the correspondingDNA-binding domains of p53 and p73. It was found that p63 variants boundthe target sequences which are bound by p53, for example, ΔNp63γ, p53,and TAp63α all yielded significant mobility shifts with three separateoligonucleotides, specifically, with a minimal p53 binding sequence, ap53 binding site in the p21 promoter WAF, and a mutant p53 binding site.Results of the assay are shown in FIG. 25.

Another aspect of the present invention features a polypeptide, of thecell regulator protein family, which functions in one of either role ofan agonist of cell-cycle regulation or an antagonist of cell-cycleregulation. In a preferred embodiment: the subject cellregulator-protein specifically binds a target DNA or protein; e.g.specifically binds a target DNA; e.g. is reasonably expected totransactivate genes involved in cell cycle arrest, such as p21;interacts with the DNA repair and synthetic machinery, such asproliferating cellular nuclear antigen, GADD 45, or proteins modulatingapoptosis. In a more preferred embodiment, the cell regulator-proteinregulates and/or modulates growth of an eukaryotic cell-cycle, e.g. amammalian cell-cycle, e.g., a human cell-cycle; the cell regulatorprotein inhibits cell growth of a eukaryotic cell, e.g., a human cell;the tumor suppressor-protein inhibits progression of a eukaryotic cellfrom G1 phase into S phase, e.g., inhibits progression of a mammaliancell from G1 phase into S phase, e.g., inhibits progression of a humancell from G1 phase into S phase; the cell regulator-protein suppressestumor growth, e.g. in a tumor cell, e.g. in a tumor cell having anunimpaired p53 or p63 or p53-like protein checkpoint. Yet another aspectof the present invention concerns an immunogen comprising a cellregulator-protein of the present invention, or a fragment thereof, in animmunogenic preparation, the immunogen being capable of eliciting animmune response specific for the cell regulator-protein; e.g. a humoralresponse, e.g., an antibody response; e.g. a cellular response. Thus, inone embodiment, the p-63 family of cell-regulatory proteins are involvedin the modulation of cell growth or regulate the growth phenotype of acell.

Another aspect of the present invention features recombinant cellregulator-protein, or a fragment thereof, cell regulatory protein, or afragment thereof, the full-length form of the cell regulatory genesprotein having an amino acid sequence at least 70% homologous to theamino acid sequence represented in one of SEQ ID Nos. 13-24; thepolypeptide has an amino acid sequence at least 80% homologous to theamino acid sequence represented in one of SEQ ID Nos. 13-24; thepolypeptide has an amino acid sequence at least 90% homologous to theamino acid sequence represented in one of SEQ ID Nos. 13-24; thepolypeptide has an amino acid sequence at least 95% homologous to theamino acid sequence represented in one of SEQ ID Nos. 13-24; thepolypeptide has an amino acid sequence identical to the amino acidsequence represented in one of SEQ ID Nos. 13-24. In a preferredembodiment: the fragment comprises at least 5 contiguous amino acidresidues of SEQ ID Nos. 13-24; the fragment comprises at least 20contiguous amino acid residues of SEQ ID Nos. 13-24; the fragmentcomprises at least 50 contiguous amino acid residues of SEQ ID Nos.13-24.

In yet another embodiment, the fragment includes the DNA binding domainof p63 and comprises at least 5 contiguous amino acid residues of SEQ IDNos. 13-24; the fragment includes the DNA binding domain of p63 andcomprises at least 20 contiguous amino acid residues of SEQ ID Nos.13-24; the fragment includes the DNA binding domain of p63 and comprisesat least 50 contiguous amino acid residues of SEQ ID Nos. 13-24.

In yet another embodiment, the fragment includes the core domain of p63and comprises at least 5 contiguous amino acid residues of SEQ ID Nos.13-24; the fragment includes the core domain of p63 and comprises atleast 20 contiguous amino acid residues of SEQ ID Nos. 13-24; thefragment includes the core domain of p63 and comprises at least 50contiguous amino acid residues of SEQ ID Nos. 13-24. The core domainextends from about amino acids “PQY” through about amino acid “HLLQ”,for instance, in TAp63γ, this region extends from about amino acid 70through about amino acid 409.

In a preferred embodiment, the recombinant cell regulator-proteinfunctions in one of either role of an agonist of cell-cycle regulationor an antagonist of cell-cycle regulation. In a preferred embodiment:the subject p63 protein specifically binds a target DNA or protein; e.g.specifically binds a target DNA; e.g. is reasonably expected totransactivate genes involved in cell cycle arrest, such as p21; interactwith the DNA repair and synthetic machinery, such as proliferatingcellular nuclear antigen, GADD 45, or proteins modulating apoptosis. Ina more preferred embodiment: the p63 protein regulates and/or modulatesgrowth of an eukaryotic cell-cycle, e.g. a mammalian cell-cycle, e.g., ahuman cell-cycle; the p63 protein inhibits cell growth of a eukaryoticcell, e.g., a human cell; the p63 protein inhibits progression of aeukaryotic cell from G1 phase into S phase, e.g., inhibits progressionof a mammalian cell from G1 phase into S phase, e.g., inhibitsprogression of a human cell from G1 phase into S phase; the tumorsuppressor-protein suppresses tumor growth, e.g. in a tumor cell, e.g.in a tumor cell having an unimpaired p53 or p63 or p53-like proteincheckpoint. Thus, in one embodiment, the p-63 family of cell-regulatoryproteins are involved in the modulation of cell growth or regulate thegrowth phenotype of a cell.

In yet other preferred embodiments, the recombinanttumor-suppressor-protein is a fusion protein further comprising a secondpolypeptide portion having an amino acid sequence from a proteinunrelated the protein of SEQ ID Nos. 13-24. Such fusion proteins can befunctional in a two-hybrid assay.

Another aspect of the present invention provides a substantially purenucleic acid having a nucleotide sequence which encodes a cellregulator-protein, or a fragment thereof, having an amino acid sequenceat least 70% homologous to one of SEQ ID Nos. 13-24. In a more preferredembodiment: the nucleic acid encodes a protein having an amino acidsequence at least 80% homologous to SEQ ID No. 5, more preferably atleast 90% homologous to SEQ ID No. 5, and most preferably at least 95%homologous to SEQ ID No. 5; the nucleic acid encodes a protein having anamino acid sequence at least 80% homologous to SEQ ID No. 8, morepreferably at least 90% homologous to SEQ ID No. 8, and most preferablyat least 95% homologous to SEQ ID No. 8 the nucleic acid encodes aprotein having an amino acid sequence at least 80% homologous to SEQ IDNo. 7, more preferably at least 90% homologous to SEQ ID No. 7, and mostpreferably at least 95% homologous to SEQ ID No. 7

In a preferred embodiment: the subject cell regulator-proteinspecifically binds a target DNA or protein; for example transactivategenes involved in cell cycle arrest, such as p21; interact with the DNArepair and synthetic machinery, such as proliferating cellular nuclearantigen, GADD 45, or proteins modulating apoptosis. In a more preferredembodiment: the cell regulator-protein regulates and/or modulates growthof an eukaryotic cell-cycle, e.g. a mammalian cell-cycle, e.g., a humancell-cycle; the cell regulator-protein inhibits cell growth of aeukaryotic cell, e.g., a human cell; the cell regulator-protein inhibitsprogression of a eukaryotic cell from G1 phase into S phase, e.g.,inhibits progression of a mammalian cell from G1 phase into S phase,e.g., inhibits progression of a human cell from G1 phase into S phase;the cell regulator-protein suppresses tumor growth, e.g. in a tumorcell, e.g. in a tumor cell having an unimpaired p53 or p63 or p53-likeprotein checkpoint. Thus, in one embodiment, the p-63 family ofcell-regulatory proteins are involved in the modulation of cell growthor regulate the growth phenotype of a cell.

In another embodiment, the nucleic acid hybridizes under stringentconditions to a nucleic acid probe corresponding to at least 12consecutive nucleotides of SEQ ID No. 1; more preferably to at least 20consecutive nucleotides of SEQ ID No. 1; more preferably to at least 40consecutive nucleotides of SEQ ID No. 1.

In a further embodiment, the nucleic acid hybridizes under stringentconditions to a nucleic acid probe corresponding to at least 12consecutive nucleotides of SEQ ID No. 2; more preferably to at least 20consecutive nucleotides of SEQ ID No. 2; more preferably to at least 40consecutive nucleotides of SEQ ID No. 2.

In yet a further embodiment, the nucleic acid hybridizes under stringentconditions to a nucleic acid probe corresponding to at least 12consecutive nucleotides of SEQ ID No. 3; more preferably to at least 20consecutive nucleotides of SEQ ID No. 3; more preferably to at least 40consecutive nucleotides of SEQ ID No. 3.

In yet a further embodiment, the nucleic acid hybridizes under stringentconditions to a nucleic acid probe corresponding to at least 12consecutive nucleotides of SEQ ID No. 6; more preferably to at least 20consecutive nucleotides of SEQ ID No. 6; more preferably to at least 40consecutive nucleotides of SEQ ID No. 6.

Furthermore, in certain embodiments, the cell regulator nucleic acidwill comprise a transcriptional regulatory sequence, e.g. at least oneof a transcriptional promoter or transcriptional enhancer sequence,operably linked to the cell regulator-gene sequence so as to render therecombinant cell regulator gene sequence suitable for use as anexpression vector.

The present invention also features transgenic non-human animals, e.g.mice, which either express a heterologous cell regulator-gene, e.g.derived from humans, or which mis-express their own cell regulator-gene,e.g. where p63, p53 or p73 expression is disrupted. Such a transgenicanimal can serve as an animal model for studying cellular disorderscomprising mutated or mis-expressed cell regulator alleles.

The present invention also provides a probe/primer comprising asubstantially purified oligonucleotide, wherein the oligonucleotidecomprises a region of nucleotide sequence which hybridizes understringent conditions to at least 10 consecutive nucleotides of sense orantisense sequence of one of SEQ ID Nos. 1-12, or naturally occurringmutants thereof. In preferred embodiments, the probe/primer furthercomprises a label group attached thereto and able to be detected, e.g.the label group is selected from a group consisting of radioisotopes,fluorescent compounds, enzymes, and enzyme co-factors. Such probes canbe used as a part of a diagnostic test kit for identifying transformedcells, such as for measuring a level of a p63, p73 or p53 encodingnucleic acid in a sample of cells isolated from a patient; e.g. formeasuring the mRNA level in a cell or determining whether the genomiccell regulator gene has been mutated or deleted.

The present invention also provides a method for treating an animalhaving unwanted cell growth characterized by a loss of wild-type cellregulator-protein function, comprising administering a therapeuticallyeffective amount of an agent able to transactivate genes involved incell cycle arrest. For instance, a therapeutically effective amount of ap63 variant comprising a transactivating domain, for example TAp63γ. Inone embodiment, the method comprises administering a nucleic acidconstruct encoding a cell regulator protein, e.g. a polypeptiderepresented in one of SEQ ID Nos. 13-24, under conditions wherein theconstruct is incorporated by cell regulator-deficient cells and thepolypeptide is expressed, e.g. by gene therapy techniques. In anotherembodiment, the method comprises administering a cell regulator mimetic,e.g. a peptidomimetic, which binds to and transactivates genes involvedin cell-cycle arrest.

Another aspect of the present invention provides a method of determiningif a subject, e.g. a human patient, is at risk for a disordercharacterized by unwanted cell proliferation, comprising detecting, in atissue of the subject, the presence or absence of a genetic lesioncharacterized by at least one of (i) a mutation of a gene encoding aprotein represented by one of SEQ ID Nos. 13-24, or a homolog thereof;or (ii) the mis-expression of the cell regulator-gene, e.g. the p63, p53or p73 gene. In preferred embodiments: detecting the genetic lesioncomprises ascertaining the existence of at least one of a deletion ofone or more nucleotides from said gene, an addition of one or morenucleotides to said gene, an substitution of one or more nucleotides ofsaid gene, a gross chromosomal rearrangement of said gene, a grossalteration in the level of a messenger RNA transcript of said gene, thepresence of a non-wild type splicing pattern of a messenger RNAtranscript of said gene, or a non-wild type level of said protein. Forexample, detecting the genetic lesion can comprise (i) providing aprobe/primer comprising an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequence ofone of SEQ ID Nos. 1-12, or naturally occurring mutants thereof, or 5′or 3′ flanking sequences naturally associated with the cellregulator-gene; (ii) exposing the probe/primer to nucleic acid of thetissue; and (iii) detecting, by hybridization of the probe/primer to thenucleic acid, the presence or absence of the genetic lesion; e.g.wherein detecting the lesion comprises utilizing the probe/primer todetermine the nucleotide sequence of the cell regulator-gene and,optionally, of the flanking nucleic acid sequences; e.g. whereindetecting the lesion comprises utilizing the probe/primer in apolymerase chain reaction (PCR); e.g. wherein detecting the lesioncomprises utilizing the probe/primer in a ligation chain reaction (LCR).In alternate embodiments, the level of said protein is detected in animmunoassay.

Yet another aspect of the invention pertains to a peptidomimetic whichtransactivates genes involved in cell-cycle arrest.

Like p53, p63 is believed to be a multifunctional protein that exerts avariety of effects and plays a central role in the regulation of thecell cycle. For instance, over-expression of p63, particularly p63variants comprising a transactivating domain may induce growth arrest,associated with the G0/G1 checkpoint, apoptosis occurring either throughthe G0/G1 checkpoint, or the S-phase or cell differentiation. Inparticular, p63 is implicated in the mechanism that senses damaged DNA,and controls its repair and in the induction of cell death. It ispossible that, this may be accomplished by transactivation of theproliferating cell nuclear antigen (PCNA), involved in DNA replicationand repair and the GADD-45 gene, whose product interacts with DNA.Furthermore, p63 may also bind several transcription associatedproteins, which are involved in DNA damage and repair machinery. Therole of p63 in tumorigenesis may be demonstrated by using p63 knockedout mice. Development of a high frequency of tumors in adult life wouldbe indicative that p63, like p53, functions as a cell regulator gene. Itis known in the art that p53 transactivates the pro-apoptotic gene, Bax(Miyashita and Reed 1995), in addition to an array of genes responsiveto oxidative stress. Based on these observations p53 has been implicatedin inducing cell-cycle arrest to allow for repair processes and/or theinduction of cell death in the event of unmitigated stress. It was seenthat p63 variants comprising a transactivation domain, i.e., TAp63γ,exhibited strong transcriptional activation of the p53 reporter. p63variants lacking the transactivational domain, e.g., Δp63 are implicatedin the regeneration of epithelial cells allowing proliferation of theepithelial cells.

It is interesting that the ΔN variants, or dominant negative, versionsof p63 seem to be the isotype expressed in many cancers. This may betied to the observation that many types of cancer, particularly cervicalcarcinoma, show an overexpression of chromosome 3q, where the p63 geneis located. If this chromosomal amplification results in theoverexpression of a ΔNp63 product which opposes p53 or transactivatingp63 forms, the experiments that follow show that the dominant negativevariants do in fact suppress the transactivating forms p63 and p53.Therefore, these p63 variants may be implicated in cancer biology andpossible diagnostic/prognostic applications.

Thus, in one embodiment, the p-63 family of cell-regulatory proteins areinvolved in the modulation of cell growth or regulate the growthphenotype of a cell. Because of its various roles activation of p63 mayresult in different outcomes as shown in FIG. 26.

Another aspect of the invention features related DNA and polypeptidesequences which are characterized by a particular percent homology oridentity as determined by any of various mathematical algorithms knownin the art. A number of mathematical algorithms have been developed tofind and measure homology between two DNA or polypeptide sequences. Forexample the local homology algorithm of Smith and Waterman ((1981)Advances in Applied Mathematics 2:482-89) is used in the alignmentsoftware program called “BestFit,” which is available from the GCGsoftware package (Genetics Computer Group, 575 Science Drive, Madison,Wis. 53711; World Wide Web at gcg.com). Other methods for aligningsequences include the homology alignment algorithm of Needleman andWunsch ((1970) J. Mol. Biol. 48: 443) and the similarity search methodof Pearson and Lipman ((1988) Proc. Natl. Acad. Sci. (USA) 85: 2444.These algorithms are available as computerized software such as GAP,FASTA and TFASTA in the Wisconsin Genetic Software Package Release 7.0,Genetics Computer Group, 575 Science Dr., Madison, Wis.). Alternatively,sequences can be aligned manually by inspection, and the best analysis,yielding the greatest degree of homology, is chosen.

These methods to describe the sequence relationships between two or morepolynucleotides require the analysis of certain elements of thesesequences and the defining of certain parameters with which to analyzethem. First, a “reference sequence” is a defined sequence used as abasis for a sequence comparison. A reference sequence may be a subset ofa larger sequence, for example, as a segment of a full-length cDNA orgene sequence is given in a sequence listing or may comprise a completecDNA or gene sequence. Generally, a reference sequence is at least 25nucleotides in length, frequently at least 25 nucleotides in length andoften at least 50 nucleotides in length. Since two polynucleotides mayeach comprise both a sequence that is similar between the twopolynucleotides and a sequence that is divergent between the twopolynucleotides, sequence comparisons between two or morepolynucleotides typically performed by comparing sequences of the twopolynucleotides over a “comparison window” to identify and compare localregions of sequence similarity. A comparison window, as used herein,refers to a conceptual segment of at least 20 contiguous nucleotidepositions wherein a polynucleotide sequence may be compared to areference sequence of at least 20 contiguous nucleotides and wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e. gaps) of 20 percent or less ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims. The practice of thepresent invention will employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames &S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, AlanR. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986);B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Primary Structure Alignments of p53, p73, and p63. Human p53,human p73β, human TAp63γ are presented, with residues identical to p53shaded in gray, and remaining consensus residues shaded in black (SEQ IDNos: 25, 26, 15, and 21).

FIGS. 2A-D: Genomic Origin and Diversity of p63 Isotypes. FIG. 2A:Schematic of human p63 gene structure highlighting positions of exons(coding sequences in black), the two promoters in exon one (black arrow)and exon 3′ (gray arrow), and the major post-transcriptional splicingevents which give rise to the major p63 isotypes. FIG. 2B: Domainstructure of p53, p73α and, β, and the major p63 isotypes, TAp63α, β,and γ, and ΔNp63 α, β, and y, highlighting regions involved intransactivation (TA), DNA binding, and oligomerization (oligo). Whitebox denotes 39aa N-terminal extension unique to TA*p63. Gray boxrepresents 14aa unique to ΔNp63. FIG. 2C: Sequence alignment ofN-termini of murine and human p63 including that found in TA*p63, TAp63,and ΔNp63 (SEQ ID Nos: 45, 46, and 47). FIG. 2D: Alignment andcomparison of the human p63 α, β, and y C-terminal sequences (SEQ IDNos: 48, 49, and 50).

FIGS. 3A-B: Chromosomal Localization of Human and Mouse p63 Gene. FIG.3A: Schematic of chromosome 3 showing localization of human p63 gene at3q27-29 based on fluorescence in situ hybridization with a p63 genomicPAC clone. FIG. 3B: Schematic of proximal end of mouse chromosome 16showing location of murine p63 gene, as determined by linkage analysisagainst Jackson Laboratory interspecific backcross panels BBS and BSB.Loci mapping to similar positions are presented in alphabetical order,and missing typing inferred from surrounding data where assignment wasunambiguous.

FIGS. 4A-E: Immunolocalization of p63 in Human Epithelial Tissues.Paraffin sections of normal human epithelial tissues probed withmonoclonal antibodies to p63 using an alkaline phosphatase reportersystem. FIG. 4A: p63 staining in foreskin showing nuclear localizationof p63 in basal epithelial cells. FIG. 4B: p63 localization to basalcells of ectocervical epithelium. FIG. 4C: p63 localization in basalcells of vaginal epithelium.

FIG. 4D: p63 staining of basal cells of urothelium. FIG. 4E: p63staining of epithelial cell layer below luminal cells in prostate.

FIGS. 5A-E: Tissue Distribution of p63 Isotypes. FIG. 5A: RT-PCRanalysis of total RNA prepared from various adult mouse tissues usingoligonucleotide primers designed to amplify TAp63 isotypes, revealing a˜410pb product in heart, testis, kidney/adrenal, thymus, brain, andcerebellum. FIG. 5B: RT-PCR analysis using template RNA in (A) witholigonucleotide primers designed to yield a ˜240 bp product for ΔNp63isotypes, revealing expected product in kidney/adrenal, spleen, andthymus. FIG. 5C: Gel electrophoresis of RNA used as template in RT-PCRanalyses to determine template integrity. FIG. 5D: Analysis of p63transcripts in human epithelial tissues. RT-PCR analyses of RNA fromprimary human foreskin keratinocytes, ectocervical cells, and the humancervical carcinoma cell line ME180 using oligonucleotides designed toamplify TAp63 transcripts (left panel) and ΔNp63 transcripts (rightpanel). The ME180 cells show products corresponding to both the TAp63and the ΔNp63 transcripts, while RNA from primary keratinocytes andectocervical cells yield predominantly products from ΔNp63 transcripts.FIG. 5E: Western blot of primary human foreskin keratinocytes (1° HFK),ME180 human cervical carcinoma cells (ME180), and BHK cells expressingepitope-tagged p63 isotypes (TA*p63γ, TAp63γ, ΔNp63γ, TA*p63a, TAp63a,and ΔNp63α) using the 4A4 anti-p63 monoclonal antibody. The major p63species in primary keratinocytes migrates slightly faster than theepitope-tagged ΔNp63α protein.

FIG. 6: Transactivation of p53-Reporter Genes by p63 Isotypes.Transcriptional activation of p53-reporter gene in Saos-2 cellstransfected with the indicated p53 and p63 expression constructs.Chemiluminescence signal from reporter β-galactosidase assays wereperformed and normalized for transfection efficiency using assays forco-transfected, constitutively expressed luciferase vectors. Error barsindicate standard deviation in triplicate assays.

FIGS. 7A-E: Induction of Apoptosis by p63 Isotypes. BHK cellstransfected with identical amounts of wildtype p53 (FIG. 7A) mutant p53(FIG. 7B), TAp63γ (FIG. 7C), ΔNp63γ (FIG. 7D), and ΔNp63α (E), wereprocessed for immunofluorescence after 16 hours using epitope-taggedantibodies (left panel) and Hoechst dye for DNA staining (right panel).Wildtype p53- and TAp63γ-expressing cells showed high levels ofapoptosis (arrows) despite very low protein expression, while ΔNp63γyielded high protein expression and modest levels of apoptosis. Mutantp53 and ΔNp63α showed high levels of protein expression but controllevels of apoptosis.

FIGS. 8A-B: Interactions Amongst p63 Isotypes and p53 in TransactivationAssays. FIG. 8A: Transactivation analysis in Saos-2 cells transfectedwith a constant amount of wildtype p53 expression vector, minimalp53-reporter construct, and either ΔNp63γ or ΔNp63α expression vectorsat ratios of 1:5 or 1:1 with respect to p53, as indicated. FIG. 8B:Transactivation analysis in Saos-2 cells transfected with a constantamount of TAp63γ expression vector, p53-reporter construct, and eitherΔNp63γ or ΔNp63α expression vectors at ratios of 1:5 or 1:1 with respectto TAp63γ, as indicated.

FIG. 9 represents nucleic acid and amino acid sequences represented bySEQ ID Nos.: 1 and 13 respectively.

FIG. 10 represents nucleic acid and amino acid sequences represented bySEQ ID Nos.: 2 and 14 respectively

FIG. 11 represents nucleic acid and amino acid sequences represented bySEQ ID Nos.: 3 and 15 respectively.

FIG. 12 represents nucleic acid and amino acid sequences represented bySEQ ID Nos.: 4 and 16 respectively.

FIG. 13 represents nucleic acid and amino acid sequences represented bySEQ ID Nos.: 5 and 17 respectively.

FIG. 14 represents nucleic acid and amino acid sequences represented bySEQ ID Nos.: 6 and 18 respectively.

FIG. 15 represents nucleic acid and amino acid sequences represented bySEQ ID Nos.: 7 and 19 respectively.

FIG. 16 represents nucleic acid and amino acid sequences represented bySEQ ID Nos.: 8 and 20 respectively.

FIG. 17 represents nucleic acid and amino acid sequences represented bySEQ ID Nos.: 9 and 21 respectively.

FIG. 18 represents nucleic acid and amino acid sequences represented bySEQ ID Nos.: 10 and 22 respectively.

FIG. 19 represents nucleic acid and amino acid sequences represented bySEQ ID Nos.: 11 and 23 respectively.

FIG. 20 represents nucleic acid and amino acid sequences represented bySEQ ID Nos.: 12 and 24 respectively.

FIG. 21 shows induction of transactivating version of p63 upon UVirradiation. The time course is similar, but not identical, to p53'sinduction by UV. This is the first demonstration that p63 (and inparticular, the transactivating, p53-like, version) can respond tostress signals such as UV/DNA damage. Primary human foreskinkeratinocytes were exposed to UV irradiation (300 J/m²), then harvestedat times indicated. Total RNA was prepared, and RT-PCR on equal amountsof RNA was performed using primers specific for transactivating (TA) ortruncated (ΔN) N-terminal domains of p63 (Yang et al., 1998). TA-p63 isnot detectable in untreated (unt) keratinocytes, but expression isinduced at 7 hrs after UV treatment. AN-p63 appears to remain unchangedfrom untreated cells at all time points taken.

FIG. 22 shows that p63 protein levels, while high in the basal,proliferative/regenerative layer of squamous epithelia, decreasesdramatically upon differentiation/maturation of these keratinocytes.This may implicate p63 in differentiation processes that are importantfor both oncogenesis and normal development. Primary human foreskinkeratinocytes were treated with 10% fetal bovine serum to inducedifferentiation, and harvested at times indicated. Cells were lysed inRIPA buffer, boiled in Lammeli buffer, and proteins fractionated by SDSgel electrophoresis. Immunoblotting was performed using an anti-p63mouse monoclonal antibody, 4A4, which recognizes all p63 isoformsidentified to date. p63 protein levels in keratinocytes are seen todecrease progressively upon differentiation, as compared to untreated(unt) samples.

FIG. 23 shows p63 RNA expression in some human cancer cell lines, mostlycervical carcinoma. RT-PCR was performed on total RNA from several humantumor cell lines (all are cervical carcinoma cell lines except U20S,which is a human osteocarcinoma line) using primers specific fortransactivating (TA) or truncated (ΔN) N-terminal domains of p63 (seeFIG. 1), as indicated. Amongst these cell lines, only the ME180 controlcell line showed a TA-p63 transcript. Instead, a majority showedexpression of ΔN-p63, which has been demonstrated to act as a dominantnegative protein towards both the tumor suppressor p53, as well astransactivating isotypes of p63.

FIG. 24 just shows p63 RNA expression in some human breast cancer celllines. It is useful to note that p63 is expressed in these cancers. ΔNversions also strongly express in some of these lines. RT-PCR wasperformed on total RNA from breast cancer cell lines using primersspecific for transactivating (TA) or truncated (ΔN) N-terminal domainsof p63 (see FIG. 1), as indicated. p63 was detected in a majority ofcell lines tested, with some specimens containing both transcriptisotypes.

FIG. 25 shows the results of an electrophoretic shift assay.Electrophoretic mobility shift assays (EMSA) were performed using threeseparate, ³²P radiolabeled oligonucleotides: a minimal p53 bindingsequence site (PG), a p53 binding site in the p2 1 promoter (WAF), and amutant p53 binding site (MG, *Kern et al., 1992) with lysates of 293human kidney cells transfected with p53, ΔNp63γ, TAp63α, and greenfluorescent protein (GFP). p53, ΔNp63γ, and TAp63α lysates all yieldedsignificant mobility shifts of both PG and WAF oligonucleotides(highlighted by arrows), while the GFP protein, included as negativecontrol, failed to display a similar shift. None of the lysates showed ashift of the control, non-p53 binding oligonucleotide, MO, thusdemonstrating the specificity of p53, ΔNp63γ, and TAp63α interactionswith the p53-binding sites.

FIG. 26 schematically depicts outcomes of p63 activation.

DETAILED DESCRIPTION

1. General

The present invention concerns the discovery of a new family of cellregulatory proteins, referred to herein as the p63 family of proteins,which demonstrate certain sequence identity to known tumor suppressorproteins p53 and p73. The p63 proteins may generally be represented bythe general formula: X-Y-Z, wherein X represents the N-termini of theproteins, e.g. a ΔN, a TA*, or TA polypeptide sequence (infra), Yrepresents the core domain of the protein, and Z represents the Ctermini, e.g., the α, β, or γ polypeptide sequences (infra). The mouseand human p63 were identified by using a novel PCR based strategy.Specifically, it was observed that the intron-exon organization wasconserved between p53 and p73, by using the known exon and intron sizesfor these genes it was possible to amplify portions of two adjacentexons and the intervening intron. The rationale being that sequencesimilarities between the exonic regions would demonstrate a relatedgene, while differences in size would indicate a novel family member. Bythis technique we identified at least one new paralog of the p53/p73/p63related family. Mouse cDNA was isolated using the RACE (5′ rapidamplification of cDNA ends) technique and the sequencing of theamplification product indicated that the amplified cDNA possessed atruncated N-terminus, i.e. the transactivation domain was absent in thisproduct. Additional splice variants of the mouse p63 were identified byscreening a cDNA library with a probe corresponding to exons 5 through 9of p63. In general, splice variants differing in the C-terminus havebeen designated as α, β, and γ forms, whereas p63 members differing inthe N-terminus are designated as the ΔN and TA forms, wherein the ΔNforms lack the transactivational domain.

The appended sequence listing, provides a list of the nucleic acid andprotein sequences that are included within the scope of this invention.

TABLE 1 Guide to p63 sequences in Sequence Listing Nucleotide Amino AcidSequence Sequence Figure hu-TAp63α SEQ ID No. 1 SEQ ID No. 13 9hu-TAp63β SEQ ID No. 2 SEQ ID No. 14 10 hu-TAp63γ SEQ ID No. 3 SEQ IDNo. 15 11 hu-ΔNp63α SEQ ID No. 4 SEQ ID No. 16 12 hu-ΔNp63β SEQ ID No. 5SEQ ID No. 17 13 hu-ΔNp63γ SEQ ID No. 6 SEQ ID No. 18 14 mu-TA*p63α SEQID No. 7 SEQ ID No. 19 15 mu-TA*p63β SEQ ID No. 8 SEQ ID No. 20 16mu-TA*p63γ SEQ ID No. 9 SEQ ID No. 21 17 mu-ΔNp63α SEQ ID No. 10 SEQ IDNo. 22 18 mu-ΔNp63β SEQ ID No. 11 SEQ ID No. 23 19 mu-ΔNp63γ SEQ ID No.12 SEQ ID No. 24 20

By fluorescence in situ hybridization (FISH), the human p63 gene hasbeen localized to chromosomal position 3q27-29. Early expression datasuggests that p63 is expressed at steady-state detectable levels invarious adult tissues. The p63 proteins can be divided into twoclasses—one with p53-like properties and the other lackingp53-associated functions such as transcriptional activation andapoptosis. p63 transcripts were detected in a wide range of adulttissues, including heart, testis, kidney/adrenal, spleen, thymus, andbrain, typically showing a predominance of either the TA or ΔN isotypes.Analysis of human epithelial tissues has provided further insights intoendogenous p63 expression, as immunohistochemistry with anti-p63monoclonal antibodies revealed strong and discrete labeling of thenuclei of basal cells within the epidermis, ectocervical epithelium,urothelium, and prostate epithelium, while more differentiated,suprabasal cells showed little or no labeling. The presence of p63 inbasal cell layers of epithelial tissues is significant because thesecells have an essential role in the regenerative processes of theseepithelia. Specifically, the basal cells are thought to be progenitor,or stem, populations for suprabasal layers, and as such are theproliferative components of these tissues. This finding that theseepithelial cells predominantly express ΔNp63 isotypes is consistent withgrowth-permitting requirements of proliferating cells, and may underliethe regenerative abilities of normal epithelial tissues.

It is contemplated by the present invention that the cloned p63-genesset out in the appended sequence listing, in addition to representing ainter-species family of related genes, are also each part of anintra-species family. That is, it is anticipated that other paralogs ofthe human and mouse p63 proteins exist in those animals, and orthologsof each p63 gene are conserved amongst other animals. For instance, atlow to medium stringency conditions, another transcript was observed andthis probably represents a new paralogous gene related to thep/53/p73/p63 family of genes, or may a splice variant of p63 as setforth in SEQ ID No. 1.

The p53 protein consists of 393 amino acids with various functionaldomains, evolutionarily conserved domains and regions which have beendesignated as mutational hotspots. The functional domains include: atransactivational domain (amino acids 20-50), sequence specific DNAbinding domain (amino acids 100-293) nuclear localization sequence(amino acids 316-325), and the oligomerization domain (amino acids319-360). It was found that the homology between p73 and p53 wasconsiderable within the most conserved p53 domains. Thetransactivational domain (amino acids 1-45) exhibited 29% identity, theDNA binding region 63% identity (amino acids 113-290) and theoligomerization domain 38% identity with p53 (amino acids 319-363).

Interestingly it was observed that the sequence identity between themouse and human p63 sequences was considerably high, both at thenucleotide and protein levels, specifically, the mouse and human p63beta forms exhibit 90.8% identity at the DNA level and 98.6% identity atthe protein level, the alpha forms showed 83.4% at the DNA level and97.8% at the protein level. The sequence identity between p63 and p73alpha form is about 57.4% identity and the p63 and p73 beta form showsabout 69.7% identity at the protein level. p63 alpha form and p53exhibit 43.8% identity at the protein level.

Accordingly, certain aspects of the present invention relate to nucleicacids encoding p63 polypeptides, the p63 polypeptides themselves(including various fragments), antibodies immunoreactive with p63proteins, and preparations of such compositions. Moreover, the presentinvention provides diagnostic and therapeutic assays and reagents fordetecting and treating disorders involving, for example, aberrantexpression (or loss) of p63.

In addition, drug discovery assays are provided for identifying agentswhich can modulate the biological function of p63 proteins, such as byaltering the binding of p63 molecules to target proteins or DNAsequences, or other extracellular/matrix factors. Furthermore, based onthe considerable sequence identity with p53, the skilled artisan couldreasonably appreciate that the p63-family of proteins would play asignificant role as a cell regulator, a tumor suppressor, function incell cycle control various developmental processes, apoptosis, geneexpression and tumorigenesis. p63 may also be implicated inhematopoiesis, muscle wasting (e.g. cachexia) and neuronaldifferentiation (and degenerative disorders related thereto). It isknown that p53 probably exists as a tetramer and dominant negativemutants of p53, which function by overwhelming the wild-type protein andprevent it from functioning probably forms a heteromeric proteincontaining both the mutant and wild-type subunits in which the wild typesubunits are unable to function. In one aspect, the inventorsdemonstrate that the p63 protein products lacking the transactivationaldomains, for example the ΔNp63α and ΔNp63γ have dominant negativeeffects on the activity of p53. Similarly, these dominant negative formsmay exert their function by forming a heteromeric protein with eitherwild-type p53 or p63 and hence prevent the wild type protein fromfunctioning.

2. Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below.

A “p63” cell regulatory protein as referred to herein, refers toproteins that may generally be represented by the formula: X-Y-Z,wherein X represents the N-termini of the proteins, e.g. a TA, TA*, orΔN polypeptide sequence, Y represents the core domain of the protein,and Z represents the C termini, e.g., the α, β, or γ polypeptidesequences. Illustrative examples include proteins represented by SEQ IDNos. 13-24, and homologs thereof.

A “p53 protein” refers to the sequence designated by GenBank AccessionNumber K03199 and orthologs thereto.

“P73” refers to the sequences disclosed by Kaghad et al., Cell90:809-819 (1997).

The term “agonist”, as used herein, is meant to refer to an agent thatmimics or upregulates (e.g. potentiates or supplements) p63 bioactivity.A p63 agonist can be a wild-type p63 protein or derivative thereofhaving at least one bioactivity of the wild-type p63. A p63 agonist canalso be a compound that upregulates expression of a gene or whichincreases at least one bioactivity of a p63 protein. An agonist can alsobe a compound which increases the interaction of a p63 polypeptide withanother molecule, e.g, a target peptide or nucleic acid.

“Antagonist” as used herein is meant to refer to an agent thatdownregulates (e.g. suppresses or inhibits) at least one p63bioactivity. A p63 antagonist can be a compound which inhibits ordecreases the interaction between a p63 protein and another molecule,e.g., a target peptide, such as angiotensin I or a kinin. Accordingly, apreferred antagonist is a compound which inhibits or decreaseshydrolysis of a target peptide. An antagonist can also be a compoundthat downregulates expression of a p63 gene or which reduces the amountof p63 protein present.

The term “antibody” as used herein is intended to whole antibodies,e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includes fragmentsthereof which are also specifically reactive with an vertebrate, e.g.,mammalian, p63 protein. Antibodies can be fragmented using conventionaltechniques and the fragments screened for utility in the same manner asdescribed above for whole antibodies. Thus, the term includes segmentsof proteolytically-cleaved or recombinantly-prepared portions of anantibody molecule that are capable of selectively reacting with a p63protein. Nonlimiting examples of such proteolytic and/or recombinantfragments include Fab, F(ab′)2, Fab′, Fv, fragments, and single chainantibodies (scFv) containing a V[L] and/or V[H] domain joined by apeptide linker. The scFv's may be covalently or non-covalently linked toform antibodies having two or more binding sites. The subject inventionincludes polyclonal, monoclonal, or other purified preparations ofantibodies and recombinant antibodies.

The term “allele”, which is used interchangeably herein with “allelicvariant” refers to alternative forms of a gene or portions thereof.Alleles occupy the same locus or position on homologous chromosomes.When a subject has two identical alleles of a gene, the subject is saidto be homozygous for that gene or allele. When a subject has twodifferent alleles of a gene, the subject is said to be heterozygous forthe gene. Alleles of a specific gene can differ from each other in asingle nucleotide, or several nucleotides, and can includesubstitutions, deletions, and/or insertions of nucleotides. An allele ofa gene can also be a form of a gene containing mutations.

The term “allelic variant of a polymorphic region of an p63 gene” refersto a region of the p63 gene having one of several nucleotide sequencesfound in that region of the gene in other individuals.

The phenomenon of “apoptosis” is well known, and can be described as aprogrammed death of cells. As is known, apoptosis is contrasted with“necrosis”, a phenomenon when cells die as a result of being killed by atoxic material, or other external effect. Apoptosis involves chromaticcondensation, membrane blebbing, and fragmentation of DNA, all of whichare generally visible upon microscopic examination.

“Biological activity” or “bioactivity” or “activity” or “biologicalfunction”, which are used interchangeably, for the purposes herein meansan effector or antigenic function that is directly or indirectlyperformed by a p63 polypeptide (whether in its native or denaturedconformation), or by any subsequence thereof. Biological activitiesinclude binding to polypeptides, particularly in the formation ofhomomeric complexes or heteromeric complexes with other p53 or p73homologs, binding to other proteins or molecules; activity as a DNAbinding protein, as a transcription regulator, ability to bind damagedDNA etc. A p63 bioactivity can be modulated by directly affecting thep63 polypeptide. Alternatively, an p63 bioactivity can be altered bymodulating the level of the p63 polypeptide, such as by modulatingexpression of the p63 gene.

As used herein the term “bioactive fragment of a p63 polypeptide” refersto a fragment of a full-length p63 polypeptide, wherein the fragmentspecifically agonizes (mimics) or antagonizes (inhibits) the activity ofa wild-type p63 polypeptide. The bioactive fragment preferably is afragment capable of interacting with at least one other molecule,protein or DNA, with which a full length p63 protein can bind.

The term “an aberrant activity”, as applied to an activity of apolypeptide such as p63, refers to an activity which differs from theactivity of the wild-type or native polypeptide or which differs fromthe activity of the polypeptide present in a healthy subject. Anactivity of a polypeptide can be aberrant because it is stronger thanthe activity of its native counterpart. Alternatively, an activity canbe aberrant because it is weaker or absent relative to the activity ofits native counterpart. An aberrant activity can also be a change in theactivity; for example, an aberrant polypeptide can interact with adifferent target peptide. A cell can have an aberrant p63 activity dueto overexpression or underexpression of the gene encoding p63.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A “chimeric polypeptide” or “fusion polypeptide” is a fusion of a firstamino acid sequence encoding one of the subject p63 polypeptides with asecond amino acid sequence defining a domain (e.g. polypeptide portion)foreign to and not substantially homologous with any domain of a p63polypeptide. A chimeric polypeptide may present a foreign domain whichis found (albeit in a different polypeptide) in an organism which alsoexpresses the first polypeptide, or it may be an “interspecies”,“intergenic”, etc. fusion of polypeptide structures expressed bydifferent kinds of organisms. In general, a fusion polypeptide can berepresented by the general formula (X)_(n)—(Y)_(m)-(Z)_(n), wherein Yrepresents a portion of the p63 polypeptide, and X and Z are eachindependently absent or represent amino acid sequences which are notrelated to the native p63 sequence found in an organism, or which arenot found as a polypeptide chain contiguous with the p63 sequence, wherem is an integer greater than or equal to one, and each occurrence of nis, independently, 0 or an integer greater than or equal to 1 (n and mare preferably no greater than 5 or 10).

The term “nucleotide sequence complementary to the nucleotide sequenceset forth in SEQ ID NO. x” refers to the nucleotide sequence of thecomplementary strand of a nucleic acid strand having SEQ ID NO. x. Theterm “complementary strand” is used herein interchangeably with the term“complement”. The complement of a nucleic acid strand can be thecomplement of a coding strand or the complement of a non-coding strand

A “delivery complex” shall mean a targeting means (e.g. a molecule thatresults in higher affinity binding of a gene, protein, polypeptide orpeptide to a target cell surface and/or increased cellular or nuclearuptake by a target cell). Examples of targeting means include: sterols(e.g. cholesterol), lipids (e.g. a cationic lipid, virosome orliposome), viruses (e.g. adenovirus, adeno-associated virus, andretrovirus) or target cell specific binding agents (e.g. ligandsrecognized by target cell specific receptors). Preferred complexes aresufficiently stable in vivo to prevent significant uncoupling prior tointernalization by the target cell. However, the complex is cleavableunder appropriate conditions within the cell so that the gene, protein,polypeptide or peptide is released in a functional form.

As is well known, genes or a particular polypeptide may exist in singleor multiple copies within the genome of an individual. Such duplicategenes may be identical or may have certain modifications, includingnucleotide substitutions, additions or deletions, which all still codefor polypeptides having substantially the same activity. The term “DNAsequence encoding a p63 polypeptide” may thus refer to one or more geneswithin a particular individual. Moreover, certain differences innucleotide sequences may exist between individual organisms, which arecalled alleles. Such allelic differences may or may not result indifferences in amino acid sequence of the encoded polypeptide yet stillencode a polypeptide with the same biological activity.

A disease, disorder or condition “associated with” or “characterized by”an aberrant p63 activity refers to a disease, disorder or condition in asubject which is caused by or contributed to by an aberrant p63activity.

The term “equivalent” is understood to include nucleotide sequencesencoding functionally equivalent p63 polypeptides or functionallyequivalent peptides having an activity of an p63 protein such asdescribed herein. Equivalent nucleotide sequences will include sequencesthat differ by one or more nucleotide substitutions, additions ordeletions, such as allelic variants; and will, therefore, includesequences that differ from the nucleotide sequence of the p63 gene shownin SEQ ID NOs: 1-12, due to the degeneracy of the genetic code.

As used herein, the terms “gene”, “recombinant gene” and “geneconstruct” refer to a nucleic acid comprising an open reading frameencoding a p63 polypeptide of the present invention, including both exonand (optionally) intron sequences.

A “recombinant gene” refers to nucleic acid encoding a p63 polypeptideand comprising p63-encoding exon sequences, though it may optionallyinclude intron sequences which are derived from, for example, achromosomal p63 gene or from an unrelated chromosomal gene. Exemplaryrecombinant genes encoding the subject p63 polypeptide are representedin the appended Sequence Listing. The term “intron” refers to a DNAsequence present in a given p63-gene which is not translated intoprotein and is generally found between exons.

The term “growth” or “growth state” of a cell refers to theproliferative state of a cell as well as to its differentiative state.Accordingly, the term refers to the phase of the cell cycle in which thecell is, e.g., G0, G1, G2, prophase, metaphase, or telophase, as well asto its state of differentiation, e.g., undifferetiated, partiallydifferentiated, or fully differentiated. Without wanting to be limited,differentiation of a cell is usually accompanied by a decrease in theproliferative rate of a cell.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules, withidentity being a more strict comparison. Homology and identity can eachbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare identical at that position. A degree of homology or similarity oridentity between nucleic acid sequences is a function of the number ofidentical or matching nucleotides at positions shared by the nucleicacid sequences. A degree of identity of amino acid sequences is afunction of the number of identical amino acids at positions shared bythe amino acid sequences. A degree of homology or similarity of aminoacid sequences is a function of the number of amino acids, i.e.structurally related, at positions shared by the amino acid sequences.An “unrelated” or “non-homologous” sequence shares less than 40%identity, though preferably less than 25% identity, with one of the p63sequences of the present invention.

The term “interact” as used herein is meant to include detectableinteractions (e.g. biochemical interactions) between molecules, such asinteraction between protein-protein, protein-nucleic acid, nucleicacid-nucleic acid, and protein-small molecule or nucleic acid-smallmolecule in nature.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs, or RNAs,respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject p63 polypeptides preferably includes no more than 10 kilobases(kb) of nucleic acid sequence which naturally immediately flanks the p63gene in genomic DNA, more preferably no more than 5 kb of such naturallyoccurring flanking sequences, and most preferably less than 1.5 kb ofsuch naturally occurring flanking sequence. The term isolated as usedherein also refers to a nucleic acid or peptide that is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Moreover, an “isolated nucleicacid” is meant to include nucleic acid fragments which are not naturallyoccurring as fragments and would not be found in the natural state. Theterm “isolated” is also used herein to refer to polypeptides which areisolated from other cellular proteins and is meant to encompass bothpurified and recombinant polypeptides.

The term “modulation” as used herein refers to both upregulation (i.e.,activation or stimulation (e.g., by agonizing or potentiating)) anddownregulation (i.e. inhibition or suppression (e.g., by antagonizing,decreasing or inhibiting)).

The term “mutated gene” refers to an allelic form of a gene, which iscapable of altering the phenotype of a subject having the mutated generelative to a subject which does not have the mutated gene. If a subjectmust be homozygous for this mutation to have an altered phenotype, themutation is said to be recessive. If one copy of the mutated gene issufficient to alter the genotype of the subject, the mutation is said tobe dominant. If a subject has one copy of the mutated gene and has aphenotype that is intermediate between that of a homozygous and that ofa heterozygous subject (for that gene), the mutation is said to beco-dominant.

The “non-human animals” of the invention include mammalians such asrodents, non-human primates, sheep, dog, cow, chickens, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse, though transgenicamphibians, such as members of the Xenopus genus, and transgenicchickens can also provide important tools for understanding andidentifying agents which can affect, for example, embryogenesis andtissue formation. The term “chimeric animal” is used herein to refer toanimals in which the recombinant gene is found, or in which therecombinant gene is expressed in some but not all cells of the animal.The term “tissue-specific chimeric animal” indicates that one of therecombinant p63 gene sis present and/or expressed or disrupted in sometissues but not others.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

The term “polymorphism” refers to the coexistence of more than one formof a gene or portion (e.g., allelic variant) thereof. A portion of agene of which there are at least two different forms, i.e., twodifferent nucleotide sequences, is referred to as a “polymorphic regionof a gene”. A polymorphic region can be a single nucleotide, theidentity of which differs in different alleles. A polymorphic region canalso be several nucleotides long.

A “polymorphic gene” refers to a gene having at least one polymorphicregion.

As used herein, the term “promoter” means a DNA sequence that regulatesexpression of a selected DNA sequence operably linked to the promoter,and which effects expression of the selected DNA sequence in cells. Theterm encompasses “tissue specific” promoters, i.e. promoters, whicheffect expression of the selected DNA sequence only in specific cells(e.g. cells of a specific tissue). The term also covers so-called“leaky” promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well. The termalso encompasses non-tissue specific promoters and promoters thatconstitutively express or that are inducible (i.e. expression levels canbe controlled).

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

The term “recombinant protein” refers to a polypeptide of the presentinvention which is produced by recombinant DNA techniques, whereingenerally, DNA encoding a p63 polypeptide is inserted into a suitableexpression vector which is in turn used to transform a host cell toproduce the heterologous protein. Moreover, the phrase “derived from”,with respect to a recombinant p63 gene, is meant to include within themeaning of “recombinant protein” those proteins having an amino acidsequence of a native p63 polypeptide, or an amino acid sequence similarthereto which is generated by mutations including substitutions anddeletions (including truncation) of a naturally occurring form of thepolypeptide.

“Small molecule” as used herein, is meant to refer to a composition,which has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be nucleic acids, peptides,polypeptides, peptidomimetics, carbohydrates, lipids or other organic(carbon containing) or inorganic molecules. Many pharmaceuticalcompanies have extensive libraries of chemical and/or biologicalmixtures, often fungal, bacterial, or algal extracts, which can bescreened with any of the assays of the invention to identify compoundsthat modulate a p63 bioactivity.

As used herein, the term “specifically hybridizes” or “specificallydetects” refers to the ability of a nucleic acid molecule of theinvention to hybridize to at least approximately 6, 12, 15, 20, 30, 50,100, 150, 200, 300, 350, 400 or 425 contiguous nucleotides of a p63gene, such as designated in any one of SEQ ID Nos: 1-12, or a sequencecomplementary thereto, or naturally occurring mutants thereof, such thatit has less than 15%, preferably less than 10%, and more preferably lessthan 5% background hybridization to a cellular nucleic acid (e.g. mRNAor genomic DNA) encoding a protein other than a p63 protein, as definedherein. In preferred embodiments, the oligonucleotide probe detects onlya p63 gene, e.g., it does not substantially hybridize to transcriptsencoding either p53 or p73, or complements thereof.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. Inpreferred embodiments, transcription of one of the p63 genes is underthe control of a promoter sequence (or other transcriptional regulatorysequence) which controls the expression of the recombinant gene in acell-type in which expression is intended. It will also be understoodthat the recombinant gene can be under the control of transcriptionalregulatory sequences which are the same or which are different fromthose sequences which control transcription of the naturally-occurringforms of p63 polypeptide.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., via an expression vector, into a recipient cell bynucleic acid-mediated gene transfer.

“Transformation”, as used herein, refers to a process in which a cell'sgenotype is changed as a result of the cellular uptake of exogenous DNAor RNA, and, for example, the transformed cell expresses a recombinantform of a p63 polypeptide or, in the case of anti-sense expression fromthe transferred gene, the expression of a naturally-occurring form ofthe p63 polypeptide is disrupted.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., one of the p63 polypeptides, or an antisense transcriptthereto) which has been introduced into a cell. A transgene could bepartly or entirely heterologous, i.e., foreign, to the transgenic animalor cell into which it is introduced, or, is homologous to an endogenousgene of the transgenic animal or cell into which it is introduced, butwhich is designed to be inserted, or is inserted, into the animal'sgenome in such a way as to alter the genome of the cell into which it isinserted (e.g., it is inserted at a location which differs from that ofthe natural gene or its insertion results in a knockout). A transgenecan also be present in a cell in the form of an episome. A transgene caninclude one or more transcriptional regulatory sequences and any othernucleic acid, such as introns, that may be necessary for optimalexpression of a selected nucleic acid.

A “transgenic animal” refers to any animal, preferably a non-humanmammal, bird or an amphibian, in which one or more of the cells of theanimal contain heterologous nucleic acid introduced by way of humanintervention, such as by transgenic techniques well known in the art.The nucleic acid is introduced into the cell, directly or indirectly byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by microinjection or by infection with arecombinant virus. The term genetic manipulation does not includeclassical cross-breeding, or in vitro fertilization, but rather isdirected to the introduction of a recombinant DNA molecule. Thismolecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of the p63 polypeptide, e.g. either agonistic orantagonistic forms. However, transgenic animals in which the recombinantp63 gene is silent are also contemplated, as for example, the FLP or CRErecombinase dependent constructs described below. Moreover, “transgenicanimal” also includes those recombinant animals in which gene disruptionof one or more p63 genes is caused by human intervention, including bothrecombination and antisense techniques.

The term “treating” as used herein is intended to encompass curing aswell as ameliorating at least one symptom of the condition or disease.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of “plasmids” which refer generally tocircular double stranded DNA loops which, in their vector form are notbound to the chromosome. In the present specification, “plasmid” and“vector” are used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

The term “wild-type allele” refers to an allele of a gene which, whenpresent in two copies in a subject results in a wild-type phenotype.There can be several different wild-type alleles of a specific gene,since certain nucleotide changes in a gene may not affect the phenotypeof a subject having two copies of the gene with the nucleotide changes.

3. Nucleic Acids of the Present Invention

As described below, one aspect of the invention pertains to isolatednucleic acids comprising a nucleotide sequence encoding p63polypeptides, variants and/or equivalents of such nucleic acids.

Preferred nucleic acids including coding sequences from vertebrate p63gene, especially a mammalian p63 gene. Regardless of the species,particularly preferred p63 nucleic acids encode polypeptides that are atleast 70%, 75%, 80%, 90%, 95%, 97%, or 98% similar to an amino acidsequence of a vertebrate p63 protein. In one embodiment, the nucleicacid is a cDNA encoding a polypeptide having at least one bio-activityof the subject p63 polypeptide. Preferably, the nucleic acid includesall or a portion of the nucleotide sequence corresponding to the nucleicacid of SEQ ID Nos 1-12.

Still other preferred nucleic acids of the present invention encode ap63 polypeptide which is comprised of at least 2, 5, 10, 25, 50, 100,150 or 200 contiguous amino acid residues. For example, preferrednucleic acid molecules for use as probes/primer or antisense molecules(i.e. noncoding nucleic acid molecules) can comprise at least about 6,12, 20, 30, 50, 60, 70, 80, 90 or 100 base pairs in length, whereascoding nucleic acid molecules can comprise about 50, 60, 70, 80, 90, or100 base pairs.

In yet another embodiment, the fragment includes the DNA binding domainof p63 and comprises at least 5 contiguous amino acid residues of SEQ IDNos. 13-24; the fragment includes the DNA binding domain of p63 andcomprises at least 20 contiguous amino acid residues of SEQ ID Nos.13-24; the fragment includes the DNA binding domain of p63 and comprisesat least 50 contiguous amino acid residues of SEQ ID Nos. 13-24.

Another aspect of the invention provides a nucleic acid which hybridizesunder low, medium, or high stringency conditions to a nucleic acidsequences represented by SEQ ID NOs: 1, 2, 3, or 4. Appropriatestringency conditions which promote DNA hybridization, for example, 6.0×sodium chloride/sodium citrate (SSC) at about 45° C., followed by a washof 2.0×SSC at 50° C., are known to those skilled in the art or can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-12.3.6. For example, the salt concentration in the washstep can be selected from a low stringency of about 2.0×SSC at 50° C. toa high stringency of about 0.2×SSC at 50° C. In addition, thetemperature in the wash step can be increased from low stringencyconditions at room temperature, about 22° C., to high stringencyconditions at about 65° C. Both temperature and salt may be varied, ortemperature of salt concentration may be held constant while the othervariable is changed. In a preferred embodiment, a p63 nucleic acid ofthe present invention will bind to one of SEQ ID NOs 1-12 undermoderately stringent conditions, for example at about 2.0×SSC and about40° C. In a particularly preferred embodiment, a p63 nucleic acid of thepresent invention will bind to one of SEQ ID NOs: 1-12 under highstringency conditions.

Preferred nucleic acids have a sequence at least 70%, and morepreferably 80% identical and more preferably 90% and even morepreferably at least 95% identical to an amino acid sequence of a p63gene, e.g., such as a sequence shown in one of SEQ ID NOS: 13-24.Nucleic acids at least 90%, more preferably 95%, and most preferably atleast about 98-99% identical with a nucleic sequence represented in oneof SEQ ID NOS: 1-12 are of course also within the scope of theinvention. In preferred embodiments, the nucleic acid is mammalian andin particularly preferred embodiments, includes all or a portion of thenucleotide sequence corresponding to the coding region of one of SEQ IDNOs: 1-12.

Nucleic acids having a sequence that differs from the nucleotidesequences shown in one of SEQ ID NOs: 1-12 due to degeneracy in thegenetic code are also within the scope of the invention. Such nucleicacids encode functionally equivalent peptides (i.e., a peptide having abiological activity of a p63 polypeptide) but differ in sequence fromthe sequence shown in the sequence listing due to degeneracy in thegenetic code. For example, a number of amino acids are designated bymore than one triplet. Codons that specify the same amino acid, orsynonyms (for example, CAU and CAC each encode histidine) may result in“silent” mutations which do not affect the amino acid sequence of a p63polypeptide. However, it is expected that DNA sequence polymorphismsthat do lead to changes in the amino acid sequences of the subject p63polypeptides will exist among mammals. One skilled in the art willappreciate that these variations in one or more nucleotides (e.g., up toabout 3-5% of the nucleotides) of the nucleic acids encodingpolypeptides having an activity of a p63 polypeptide may exist amongindividuals of a given species due to natural allelic variation.

Also within the scope of the invention are nucleic acids encodingsplicing variants of p63 proteins or natural homologs of p63 proteinswhich consist essentially of one of the two units of p63. Such homologscan be cloned by hybridization or PCR, as further described herein.

The polynucleotide sequence may also encode for a leader sequence, e.g.,the natural leader sequence or a heterologous leader sequence. Forexample, the desired DNA sequence may be fused in the same reading frameto a DNA sequence which aids in expression and secretion of thepolypeptide from the host cell, for example, a leader sequence whichfunctions as a secretory sequence for controlling transport of thepolypeptide from the cell. The protein having a leader sequence is apreprotein and may have the leader sequence cleaved by the host cell toform the mature form of the protein.

The polynucleotide of the present invention may also be fused in frameto a marker sequence, also referred to herein as “Tag sequence” encodinga “Tag peptide”, which allows for marking and/or purification of thepolypeptide of the present invention. In a preferred embodiment, themarker sequence is a hexahistidine tag, e.g., supplied by a PQE-9vector. Numerous other Tag peptides are available commercially. Otherfrequently used Tags include myc-epitopes (e.g., see Ellison et al.(1991) J Biol Chem 266:21150-21157) which includes a 10-residue sequencefrom c-myc, the pFLAG system (International Biotechnologies, Inc.), thepEZZ-protein A system (Pharmacia, NJ), and a 16 amino acid portion ofthe Haemophilus influenza hemagglutinin protein. Furthermore, anypolypeptide can be used as a Tag so long as a reagent, e.g., an antibodyinteracting specifically with the Tag polypeptide is available or can beprepared or identified.

As indicated by the examples set out below, p63 protein-encoding nucleicacids can be obtained from mRNA present in any of a number of eukaryoticcells, e.g., and is preferably obtained from metazoan cells, morepreferably from vertebrate cells and even more preferably from mammaliancells. It should also be possible to obtain nucleic acids encoding p63polypeptides of the present invention from genomic DNA from both adultsand embryos. For example, a gene encoding a p63 protein can be clonedfrom either a cDNA or a genomic library in accordance with protocolsdescribed herein, as well as those generally known to persons skilled inthe art. cDNA encoding a p63 protein can be obtained by isolating totalmRNA from a cell, e.g., a vertebrate cell, a mammalian cell, or a humancell, including embryonic cells. Double stranded cDNAs can then beprepared from the total mRNA, and subsequently inserted into a suitableplasmid or bacteriophage vector using any one of a number of knowntechniques. The gene encoding a p63 protein can also be cloned usingestablished polymerase chain reaction techniques in accordance with thenucleotide sequence information provided by the invention. A preferrednucleic acid is a cDNA represented by a sequence selected from the groupconsisting of SEQ ID NOs: 1-12.

Preferred nucleic acids encode a vertebrate p63 polypeptide comprisingan amino acid sequence at least 80% identical, more preferably 90%identical and most preferably 95% identical with an amino acid sequencecontained in any of SEQ ID Nos: 13-24. Nucleic acids which encodepolypeptides at least about 90%, more preferably at least about 95%, andmost preferably at least about 98-99% homology with an amino acidsequence represented in SEQ ID No: 13-24 are also within the scope ofthe invention. In one embodiment, the nucleic acid is a cDNA encoding apeptide having at least one activity of the subject vertebrate p63polypeptide. Preferably, the nucleic acid includes all or a portion ofthe nucleotide sequence corresponding to the coding region of SEQ IDNos: 1-12.

Preferred nucleic acids encode a bioactive fragment of a vertebrate p63polypeptide comprising an amino acid sequence at least 80% identical oridentical, more preferably 90% identical or identical and mostpreferably 95% identical or identical with an amino acid sequenceselected from the group consisting of SEQ ID No: 13-24. For instance,these bioactive fragments may include the DNA binding domains,transactivation domains, oligomerization domain, etc. Nucleic acidswhich encode polypeptides which are at least about 90%, more preferablyat least about 95%, and most preferably these at least about 98-99%homologous or identical, with an amino acid sequence represented in SEQID No: 13-24 are also within the scope of the invention.

Preferred bioactive fragments of p63 polypeptides include polypeptideshaving one or more of the following biological activities: activity as atumor suppressor, functions in cell cycle control of variousdevelopmental processes, apoptosis, gene expression, modulation ofproliferation and differentiation, and tumorigenesis. Assays fordetermining whether given homolog of a p63 exhibits these or otherbiological activities are known in the art and are further describedherein.

In yet another embodiment, the fragment includes the DNA binding domainof p63 and comprises at least 5 contiguous amino acid residues of SEQ IDNos. 13-24; the fragment includes the DNA binding domain of p63 andcomprises at least 20 contiguous amino acid residues of SEQ ID Nos.13-24; the fragment includes the DNA binding domain of p63 and comprisesat least 50 contiguous amino acid residues of SEQ ID Nos. 13-24.

In yet another embodiment, the fragment includes the core domain of p63and comprises at least 5 contiguous amino acid residues of SEQ ID Nos.13-24; the fragment includes the core domain of p63 and comprises atleast 20 contiguous amino acid residues of SEQ ID Nos. 13-24; thefragment includes the core domain of p63 and comprises at least 50contiguous amino acid residues of SEQ ID Nos. 13-24.

3.1. Probes and Primers

The nucleotide sequences determined from the cloning of p63 genes frommammalian organisms will further allow for the generation of probes andprimers designed for identifying and/or cloning p63 homologs in othercell types, e.g., from other tissues, as well as p63 homologs from othermammalian organisms. For instance, the present invention also provides aprobe/primer comprising a substantially purified oligonucleotide, whicholigonucleotide comprising a nucleotide sequence that hybridizes understringent conditions to at least approximately 12, preferably 25, morepreferably 40, 50 or 75 consecutive nucleotides of sense or anti-sensesequence selected from the group consisting of SEQ ID No: 1-12 ornaturally occurring mutants thereof. For instance, primers based on thenucleic acid represented in SEQ ID NOs: 1-12 can be used in PCRreactions to clone p63 homologs.

In yet another embodiment, the invention provides probes/primerscomprising a substantially purified oligonucleotide comprising anucleotide sequence that hybridizes under moderately stringentconditions to at least approximately 12, 16, 25, 40, 50 or 75consecutive nucleotides sense or antisense sequence selected from thegroup consisting of SEQ ID NOS. 1-12, or naturally occurring mutantsthereof.

In particular, these probes are useful because they provide a method fordetecting mutations in tumor suppressor genes such as p63, p73, p53 orRb etc. Nucleic acid probes which are complementary to the wild-type p63and can form mismatches with mutant p63 genes are provided, which allowfor detection by enzymatic or chemical cleavage or by shifts inelectrophonetic mobility.

Likewise, probes based on the subject p63 sequences can be used todetect transcripts or genomic sequences encoding the same or homologousproteins, for use, e.g, in prognostic or diagnostic assays. In preferredembodiments, the probe further comprises a label group attached theretoand able to be detected, e.g., the label group is selected from amongstradioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.

3.2 Antisense, Ribozyme and Triplex Techniques

Another aspect of the invention relates to the use of the isolatednucleic acid in “antisense” therapy. As used herein, “antisense” therapyrefers to administration or in situ generation of oligonucleotidemolecules or their derivatives which specifically hybridize (e.g., bind)under cellular conditions, with the cellular mRNA and/or genomic DNAencoding one or more of the subject p63 proteins so as to inhibitexpression of that protein, e.g., by inhibiting transcription and/ortranslation. The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to the range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes a p63 protein. Alternatively, the antisenseconstruct is an oligonucleotide probe which is generated ex vivo andwhich, when introduced into the cell causes inhibition of expression byhybridizing with the mRNA and/or genomic sequences of a p63 gene. Sucholigonucleotide probes are preferably modified oligonucleotides whichare resistant to endogenous nucleases, e.g., exonucleases and/orendonucleases, and are therefore stable in vivo. Exemplary nucleic acidmolecules for use as antisense oligonucleotides are phosphoramidate,phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat.Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, generalapproaches to constructing oligomers useful in antisense therapy havebeen reviewed, for example, by Van der Krol et al. (1988) BioTechniques6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668. With respectto antisense DNA, oligodeoxyribonucleotides derived from the translationinitiation site, e.g., between the −10 and +10 regions of thep63nucleotide sequence of interest, are preferred.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to p63 mRNA. The antisenseoligonucleotides will bind to the p63 mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired. In the case of double-stranded antisense nucleic acids, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acid.Generally, the longer the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the mRNA, e.g.,the 5′ untranslated sequence up to and including the AUG initiationcodon, should work most efficiently at inhibiting translation. However,sequences complementary to the 3′ untranslated sequences of mRNAs haverecently been shown to be effective at inhibiting translation of mRNAsas well. (Wagner, R. 1994. Nature 372:333). Therefore, oligonucleotidescomplementary to either the 5′ or 3′ untranslated, non-coding regions ofa p63 gene could be used in an antisense approach to inhibit translationof endogenous p63 mRNA. Oligonucleotides complementary to the 5′untranslated region of the mRNA should include the complement of the AUGstart codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could also beused in accordance with the invention. Whether designed to hybridize tothe 5′, 3′ or coding region of p63 mRNA, antisense nucleic acids shouldbe at least six nucleotides in length, and are preferably less thatabout 100 and more preferably less than about 50, 25, 17 or 10nucleotides in length.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to quantitate the ability of the antisenseoligonucleotide to inhibit gene expression. It is preferred that thesestudies utilize controls that distinguish between antisense geneinhibition and nonspecific biological effects of oligonucleotides. It isalso preferred that these studies compare levels of the target RNA orprotein with that of an internal control RNA or protein. Additionally,it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. the oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors), or agents facilitating transport across the cell membrane(see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652;PCT Publication No. WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. WO9/10134, publishedApr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g.,Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents.(See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc. The antisenseoligonucleotide may comprise at least one modified base moiety which isselected from the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxytiethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

The antisense oligonucleotide can also contain a neutral peptide-likebackbone. Such molecules are termed peptide nucleic acid (PNA)-oligomersand are described, e.g., in Perry-O'Keefe et al. (1996) Proc. Natl.Acad. Sci. U.S.A. 93:14670 and in Eglom et al. (1993) Nature 365:566.One advantage of PNA oligomers is their capability to bind tocomplementary DNA essentially independently from the ionic strength ofthe medium due to the neutral backbone of the DNA. In yet anotherembodiment, the antisense oligonucleotide comprises at least onemodified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet a further embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-12148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

While antisense nucleotides complementary to the p63 coding regionsequence can be used, those complementary to the transcribeduntranslated region and to the region comprising the initiatingmethionine are most preferred.

The antisense molecules can be delivered to cells which express p63 invivo. A number of methods have been developed for delivering antisenseDNA or RNA to cells; e.g., antisense molecules can be injected directlyinto the tissue site, or modified antisense molecules, designed totarget the desired cells (e.g., antisense linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systematically.

However, it is often difficult to achieve intracellular concentrationsof the antisense sufficient to suppress translation on endogenous mRNAs.Therefore a preferred approach utilizes a recombinant DNA construct inwhich the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect target cells in the patient will result in the transcriptionof sufficient amounts of single stranded RNAs that will formcomplementary base pairs with the endogenous p63 transcripts and therebyprevent translation of the p63 mRNA. For example, a vector can beintroduced in vivo such that it is taken up by a cell and directs thetranscription of an antisense RNA. Such a vector can remain episomal orbecome chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al, 1982, Nature 296:39-42), etc. Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct which can be introduced directly into the tissue site; e.g.,the choroid plexus or hypothalamus. Alternatively, viral vectors can beused which selectively infect the desired tissue; (e.g., for brain,herpesvirus vectors may be used), in which case administration may beaccomplished by another route (e.g., systematically).

Ribozyme molecules designed to catalytically cleave p63 mRNA transcriptscan also be used to prevent translation of p63 mRNA and expression ofp63 (See, e.g., PCT International Publication WO90/11364, published Oct.4, 1990; Sarver et al., 1990, Science 247:1222-1225 and U.S. Pat. No.5,093,246). While ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy p63 mRNAs, the use ofhammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.There are a number of potential hammerhead ribozyme cleavage siteswithin the nucleotide sequence of human p63 cDNA (FIG. 1). Preferablythe ribozyme is engineered so that the cleavage recognition site islocated near the 5′ end of the p63 mRNA; i.e., to increase efficiencyand minimize the intracellular accumulation of non-functional mRNAtranscripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; published International patent application No. WO88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in a p63 gene.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells which express the p63 gene in vivo. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous p63 messages andinhibit translation. Because ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

Endogenous p63 gene expression can also be reduced by inactivating or“knocking out” the p63 gene or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., 1985, Nature 317:230-234;Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al., 1989 Cell5:313-321; each of which is incorporated by reference herein in itsentirety). For example, a mutant, non-functional p63 (or a completelyunrelated DNA sequence) flanked by DNA homologous to the endogenous p63gene (either the coding regions or regulatory regions of the p63 gene)can be used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express p63 in vivo.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the p63 gene. Such approaches areparticularly suited in the agricultural field where modifications to ES(embryonic stem) cells can be used to generate animal offspring with aninactive p63 (e.g., see Thomas & Capecchi 1987 and Thompson 1989,supra). However this approach can be adapted for use in humans providedthe recombinant DNA constructs are directly administered or targeted tothe required site in vivo using appropriate viral vectors, e.g., herpesvirus vectors for delivery to brain tissue; e.g., the hypothalamusand/or choroid plexus.

Alternatively, endogenous p63 gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the p63 gene (i.e., the p63 promoter and/or enhancers) to formtriple helical structures that prevent transcription of the p63 gene intarget cells in the body. (See generally, Helene, C. 1991, AnticancerDrug Des., 6(6):569-84; Helene, C., et al., 1992, Ann, N.Y. Accad. Sci.,660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15).

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription are preferably single stranded and composedof deoxyribonucleotides. The base composition of these oligonucleotidesshould promote triple helix formation via Hoogsteen base pairing rules,which generally require sizable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′,3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Antisense RNA and DNA, ribozyme, and triple helix molecules of theinvention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Moreover, various well-known modifications to nucleic acid molecules maybe introduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone.

3.3. Vectors Encoding p63 Proteins and p63 Expressing Cells

The invention further provides plasmids and vectors encoding an p63protein, which can be used to express an p63 protein in a host cell. Thehost cell may be any prokaryotic or eukaryotic cell. Thus, a nucleotidesequence derived from the cloning of mammalian p63 proteins, encodingall or a selected portion of the full-length protein, can be used toproduce a recombinant form of an p63 polypeptide via microbial oreukaryotic cellular processes. Ligating the polynucleotide sequence intoa gene construct, such as an expression vector, and transforming ortransfecting into hosts, either eukaryotic (yeast, avian, insect ormammalian) or prokaryotic (bacterial cells), are standard procedureswell known in the art.

Vectors that allow expression of a nucleic acid in a cell are referredto as expression vectors. Typically, expression vectors used forexpressing an p63 protein contain a nucleic acid encoding an p63polypeptide, operably linked to at least one transcriptional regulatorysequence. Regulatory sequences are art-recognized and are selected todirect expression of the subject p63 proteins. Transcriptionalregulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). In one embodiment, the expression vector includes a recombinantgene encoding a peptide having an agonistic activity of a subject p63polypeptide, or alternatively, encoding a peptide which is anantagonistic form of an p63 protein.

Suitable vectors for the expression of a p63 polypeptide includeplasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, a p63 polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the p63 genes represented in SEQ ID NOs: 1or 3.

The preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAIo/neo, pRc/CMV, pSV2gpt,pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derivedvectors are examples of mammalian expression vectors suitable fortransfection of eukaryotic cells. Some of these vectors are modifiedwith sequences from bacterial plasmids, such as pBR322, to facilitatereplication and drug resistance selection in both prokaryotic andeukaryotic cells. Alternatively, derivatives of viruses such as thebovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook,Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989)Chapters 16 and 17.

In some instances, it may be desirable to express the recombinant p63polypeptide by the use of a baculovirus expression system. Examples ofsuch baculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal containing pBlueBacIII).

When it is desirable to express only a portion of a p63 protein, such asa form lacking a portion of the N-terminus, i.e. a truncation mutantwhich lacks the signal peptide, it may be necessary to add a start codon(ATG) to the oligonucleotide fragment containing the desired sequence tobe expressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing p63 derived polypeptides in a hostwhich produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitroby use of purified MAP (e.g., procedure of Miller et al., supra).

Moreover, the gene constructs of the present invention can also be usedas part of a gene therapy protocol to deliver nucleic acids encodingeither an agonistic or antagonistic form of one of the subject p63proteins. Thus, another aspect of the invention features expressionvectors for in vivo or in vitro transfection and expression of a p63polypeptide in particular cell types so as to reconstitute the functionof, or alternatively, abrogate the function of p63 in a tissue. Thiscould be desirable, for example, when the naturally-occurring form ofthe protein is misexpressed or the natural protein is mutated and lessactive.

In addition to viral transfer methods, non-viral methods can also beemployed to cause expression of a subject p63 polypeptide in the tissueof an animal. Most nonviral methods of gene transfer rely on normalmechanisms used by mammalian cells for the uptake and intracellulartransport of macromolecules. In preferred embodiments, non-viraltargeting means of the present invention rely on endocytic pathways forthe uptake of the subject p63 polypeptide gene by the targeted cell.Exemplary targeting means of this type include liposomal derivedsystems, poly-lysine conjugates, and artificial viral envelopes.

In other embodiments transgenic animals, described in more detail belowcould be used to produce recombinant proteins.

4. Polypeptides of the Present Invention

The present invention makes available isolated p63 polypeptides whichare isolated from, or otherwise substantially free of other cellularproteins, especially other signal transduction factors and/ortranscription factors which may normally be associated with the p63polypeptide. The term “substantially free of other cellular proteins”(also referred to herein as “contaminating proteins”) or “substantiallypure or purified preparations” are defined as encompassing preparationsof p63 polypeptides having less than about 20% (by dry weight)contaminating protein, and preferably having less than about 5%contaminating protein. Functional forms of the subject polypeptides canbe prepared, for the first time, as purified preparations by using acloned gene as described herein. Full length proteins or fragmentscorresponding to one or more particular motifs and/or domains or toarbitrary sizes, for example, at least 5, 10, 25, 50, 75 and 100, aminoacids in length are within the scope of the present invention.

In yet another embodiment, the fragment includes the core domain of p63and comprises at least 5 contiguous amino acid residues of SEQ ID Nos.13-24; the fragment includes the core domain of p63 and comprises atleast 20 contiguous amino acid residues of SEQ ID Nos. 13-24; thefragment includes the core domain of p63 and comprises at least 50contiguous amino acid residues of SEQ ID Nos. 13-24.

In yet another embodiment, the fragment includes the DNA binding domainof p63 and comprises at least 5 contiguous amino acid residues of SEQ IDNos. 13-24; the fragment includes the DNA binding of p63 and comprisesat least 20 contiguous amino acid residues of SEQ ID Nos. 13-24; thefragment includes the DNA binding of p63 and comprises at least 50contiguous amino acid residues of SEQ ID Nos. 13-24.

For example, isolated p63 polypeptides can be encoded by all or aportion of a nucleic acid sequence shown in any of SEQ ID NOS. 1-12.Isolated peptidyl portions of p63 proteins can be obtained by screeningpeptides recombinantly produced from the corresponding fragment of thenucleic acid encoding such peptides. In addition, fragments can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Forexample, a p63 polypeptide of the present invention may be arbitrarilydivided into fragments of desired length with no overlap of thefragments, or preferably divided into overlapping fragments of a desiredlength. The fragments can be produced (recombinantly or by chemicalsynthesis) and tested to identify those peptidyl fragments which canfunction as either agonists or antagonists of a wild-type (e.g.,“authentic”) p63 protein.

Another aspect of the present invention concerns recombinant forms ofthe p63 proteins. Recombinant polypeptides preferred by the presentinvention, in addition to native p63 proteins (e.g., as set forth in SEQID NO: 6), are encoded by a nucleic acid, which is at least 60%, morepreferably at least 80%, and more preferably 85%, and more preferably90%, and more preferably 95% identical to an amino acid sequencerepresented by SEQ ID Nos: 13-24 or encoded by SEQ ID NOs. 13-24.Polypeptides which are encoded by a nucleic acid that is at least about98-99% identical with the sequence of SEQ ID NOS: 1 or 3 or which are98-99% identical with the amino acid sequence set forth in SEQ ID NO: 2are also within the scope of the invention.

In a preferred embodiment, a p63 protein of the present invention is amammalian p63 protein and even more preferably a human p63 protein. In aparticularly preferred embodiment the p63 protein has an amino acidsequence as set forth in SEQ ID Nos: 13-24. In particularly preferredembodiment, the p63 protein retains p63 bioactivity. It will beunderstood that certain post-translational modifications, e.g.,phosphorylation and the like, can increase the apparent molecular weightof the p63 protein relative to the unmodified polypeptide chain.

The present invention further pertains to recombinant forms of one ofthe subject p63 polypeptides. Such recombinant p63 polypeptidespreferably are capable of functioning in one of either role of anagonist or antagonist of at least one biological activity of a wild-type(“authentic”) p63 protein of the appended sequence listing. The term“evolutionarily related to”, with respect to amino acid sequences of p63proteins, refers to both polypeptides having amino acid sequences whichhave arisen naturally, and also to mutational variants of human p63polypeptides which are derived, for example, by combinatorialmutagenesis.

In general, polypeptides referred to herein as having an activity (e.g.,are “bioactive”) of a p63 protein are defined as polypeptides whichinclude an amino acid sequence encoded by all or a portion of thenucleic acid sequences shown in one of SEQ ID NOS: 1-12 and which mimicor antagonize all or a portion of the biological/biochemical activitiesof a naturally occurring p63 protein. Preferred bioactive fragments ofp63 polypeptides include polypeptides having one or more of thefollowing biological activities: activity as a tumor suppressor,functions in cell cycle control various developmental processes,apoptosis, gene expression and tumorigenesis. Other biologicalactivities of the subject p63 proteins are described herein or will bereasonably apparent to those skilled in the art. According to thepresent invention, a polypeptide has biological activity if it is aspecific agonist or antagonist of a naturally-occurring form of a p63protein.

Assays for determining whether a compound, e.g, a protein, such as anp63 protein or variant thereof, has one or more of the above biologicalactivities are well known in the art.

In another embodiment, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide. This type of expression system can beuseful under conditions where it is desirable to produce an immunogenicfragment of a p63 protein. For example, the VP6 capsid protein ofrotavirus can be used as an immunologic carrier protein for portions ofthe p63 polypeptide, either in the monomeric form or in the form of aviral particle. The nucleic acid sequences corresponding to the portionof a subject p63 protein to which antibodies are to be raised can beincorporated into a fusion gene construct which includes codingsequences for a late vaccinia virus structural protein to produce a setof recombinant viruses expressing fusion proteins comprising p63epitopes as part of the virion. It has been demonstrated with the use ofimmunogenic fusion proteins utilizing the Hepatitis B surface antigenfusion proteins that recombinant Hepatitis B virions can be utilized inthis role as well. Similarly, chimeric constructs coding for fusionproteins containing a portion of a p63protein and the poliovirus capsidprotein can be created to enhance immunogenicity of the set ofpolypeptide antigens (see, for example, EP Publication No: 0259149; andEvans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol.62:3855; and Schlienger et al. (1992) J. Virol. 66:2).

The Multiple antigen peptide system for peptide-based immunization canalso be utilized to generate an immunogen, wherein a desired portion ofa p63 polypeptide is obtained directly from organo-chemical synthesis ofthe peptide onto an oligomeric branching lysine core (see, for example,Posnett et al. (1988) JBC 263:1719 and Nardelli et al. (1992) J.Immunol. 148:914). Antigenic determinants of p63 proteins can also beexpressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins, and accordingly, can be used in the expressionof the p63 polypeptides of the present invention. For example, p63polypeptides can be generated as glutathione-S-transferase (GST-fusion)proteins. Such GST-fusion proteins can enable easy purification of thep63 polypeptide, as for example by the use of glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant protein, canallow purification of the expressed fusion protein by affinitychromatography using a Ni²⁺ metal resin. The purification leadersequence can then be subsequently removed by treatment with enterokinaseto provide the purified protein (e.g., see Hochuli et al. (1987) J.Chromatography 411:177; and Janknecht et al. PNAS 88:8972). Techniquesfor making fusion genes are known to those skilled in the art.Essentially, the joining of various DNA fragments coding for differentpolypeptide sequences is performed in accordance with conventionaltechniques, employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed to generate a chimeric genesequence (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. John Wiley & Sons: 1992).

The present invention further pertains to methods of producing thesubject p63 polypeptides. For example, a host cell transfected with anucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. Suitable mediafor cell culture are well known in the art. The recombinant p63polypeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such peptide. In a preferred embodiment, therecombinant p63 polypeptide is a fusion protein containing a domainwhich facilitates its purification, such as GST fusion protein.

Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of one of thesubject p63 polypeptides which function in a limited capacity as one ofeither a p63 agonist (mimetic) or a p63 antagonist, in order to promoteor inhibit only a subset of the biological activities of thenaturally-occurring form of the protein. Thus, specific biologicaleffects can be elicited by treatment with a homolog of limited function,and with fewer side effects relative to treatment with agonists orantagonists which are directed to all of the biological activities ofnaturally occurring forms of p63 proteins.

Homologs of each of the subject p63 proteins can be generated bymutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologs which retainsubstantially the same, or merely a subset, of the biological activityof the p63 polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to an p63 receptor.

The recombinant p63 polypeptides of the present invention also includehomologs of the wildtype p63 proteins, such as versions of those proteinwhich are resistant to proteolytic cleavage, as for example, due tomutations which alter ubiquitination or other enzymatic targetingassociated with the protein.

p63 polypeptides may also be chemically modified to create p63derivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of p63 proteins can beprepared by linking the chemical moieties to functional groups on aminoacid sidechains of the protein or at the N-terminus or at the C-terminusof the polypeptide.

Modification of the structure of the subject p63 polypeptides can be forsuch purposes as enhancing therapeutic or prophylactic efficacy,stability (e.g., ex vivo shelf life and resistance to proteolyticdegradation), or post-translational modifications (e.g., to alterphosphorylation pattern of protein). Such modified peptides, whendesigned to retain at least one activity of the naturally-occurring formof the protein, or to produce specific antagonists thereof, areconsidered functional equivalents of the p63 polypeptides described inmore detail herein. Such modified peptides can be produced, forinstance, by amino acid substitution, deletion, or addition. Thesubstitutional variant may be a substituted conserved amino acid or asubstituted non-conserved amino acid.

For example, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (i.e. isosteric and/or isoelectricmutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (see, for example, Biochemistry, 2^(nd) ed., Ed. by L.Stryer, WH Freeman and Co.: 1981). Whether a change in the amino acidsequence of a peptide results in a functional p63 homolog (e.g.,functional in the sense that the resulting polypeptide mimics orantagonizes the wild-type form) can be readily determined by assessingthe ability of the variant peptide to produce a response in cells in afashion similar to the wild-type protein, or competitively inhibit sucha response. Polypeptides in which more than one replacement has takenplace can readily be tested in the same manner.

This invention further contemplates a method for generating sets ofcombinatorial mutants of the subject p63 proteins as well as truncationmutants, and is especially useful for identifying potential variantsequences (e.g., homologs). The purpose of screening such combinatoriallibraries is to generate, for example, novel p63 homologs which can actas either agonists or antagonist, or alternatively, possess novelactivities all together. Thus, combinatorially-derived homologs can begenerated to have an increased potency relative to a naturally occurringform of the protein.

In one embodiment, the variegated library of p63 variants is generatedby combinatorial mutagenesis at the nucleic acid level, and is encodedby a variegated gene library. For instance, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential p63 sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of p63 sequencestherein. There are many ways by which such libraries of potential p63homologs can be generated from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be carried out inan automatic DNA synthesizer, and the synthetic genes then ligated intoan appropriate expression vector. The purpose of a degenerate set ofgenes is to provide, in one mixture, all of the sequences encoding thedesired set of potential p63 sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc3^(rd) Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam:Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477. Such techniques have been employed in the directedevolution of other proteins (see, for example, Scott et al. (1990)Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin etal. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Likewise, a library of coding sequence fragments can be provided for ap63 clone in order to generate a variegated population of p63 fragmentsfor screening and subsequent selection of bioactive fragments. A varietyof techniques are known in the art for generating such libraries,including chemical synthesis. In one embodiment, a library of codingsequence fragments can be generated by (i) treating a double strandedPCR fragment of a p63 coding sequence with a nuclease under conditionswherein nicking occurs only about once per molecule; (ii) denaturing thedouble stranded DNA; (iii) renaturing the DNA to form double strandedDNA which can include sense/antisense pairs from different nickedproducts; (iv) removing single stranded portions from reformed duplexesby treatment with S1 nuclease; and (v) ligating the resulting fragmentlibrary into an expression vector. By this exemplary method, anexpression library can be derived which codes for N-terminal, C-terminaland internal fragments of various sizes.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of p63 homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of degenerate p63 sequences created bycombinatorial mutagenesis techniques. Combinatorial mutagenesis has apotential to generate very large libraries of mutant proteins, e.g., inthe order of 10²⁶ molecules. Combinatorial libraries of this size may betechnically challenging to screen even with high throughput screeningassays. To overcome this problem, a new technique has been developedrecently, recrusive ensemble mutagenesis (REM), which allows one toavoid the very high proportion of non-functional proteins in a randomlibrary and simply enhances the frequency of functional proteins, thusdecreasing the complexity required to achieve a useful sampling ofsequence space. REM is an algorithm which enhances the frequency offunctional mutants in a library when an appropriate selection orscreening method is employed (Arkin and Yourvan, 1992, PNAS USA89:7811-7815; Yourvan et al., 1992, Parallel Problem Solving fromNature, 2., In Maenner and Manderick, eds., Elsevir Publishing Co.,Amsterdam, pp. 401-410; Delgrave et al., 1993, Protein Engineering6(3):327-331).

The invention also provides for reduction of the p63 proteins togenerate mimetics, e.g., peptide or non-peptide agents, such as smallmolecules, which are able to disrupt binding of a p63 polypeptide of thepresent invention with a nucleotide, such as proteins, e.g. receptors.Thus, such mutagenic techniques as described above are also useful tomap the determinants of the p63 proteins which participate inprotein-protein interactions involved in, for example, binding of thesubject p63 polypeptide to a target peptide. To illustrate, the criticalresidues of a subject p63 polypeptide which are involved in molecularrecognition of its receptor can be determined and used to generate p63derived peptidomimetics or small molecules which competitively inhibitbinding of the authentic p63 protein with that moiety. By employing, forexample, scanning mutagenesis to map the amino acid residues of thesubject p63 proteins which are involved in binding other proteins,peptidomimetic compounds can be generated which mimic those residues ofthe p63 protein which facilitate the interaction. Such mimetics may thenbe used to interfere with the normal function of ap63 protein. Forinstance, non-hydrolyzable peptide analogs of such residues can begenerated using benzodiazepine (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gamma lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson etal. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structureand Function (Proceedings of the 9^(th) American Peptide Symposium)Pierce Chemical Co. Rockland, Ill., 1985), 1-turn dipeptide cores (Nagaiet al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem SocPerkin Trans 1:1231), and β-aminoalcohols (Gordon et al. (1985) BiochemBiophys Res Commun 126:419; and Dann et al. (1986) Biochem Biophys ResCommun 134:71)

5. Anti-p63 Antibodies and Uses Therefor

Another aspect of the invention pertains to an antibody specificallyreactive with a mammalian p63 protein, e.g., a wild-type or mutated p63protein. For example antibodies may be made as described in the appendedexamples or by using other standard protocols (See, for example,Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold SpringHarbor Press: 1988)). A mammal, such as a mouse, a hamster or rabbit canbe immunized with an immunogenic form of the peptide (e.g., a mammalianp63 polypeptide or an antigenic fragment which is capable of elicitingan antibody response, or a fusion protein as described above).

In one aspect, this invention includes monoclonal antibodies to p63 thatshow p63 is highly expressed in the basal cells of various epithelialtissues, including epidermis, ectocervical epithelium, vaginalepithelium, urothelium, and prostate epthelium, all of which representcommon sites of human carcinomas (basal cell carcinoma of skin, cervicalcarcinoma with and without human papilloma virus association, bladderand urothelial carcinoma, and prostate carcinoma). Therefore, in oneembodiment this invention provides a diagnostic tool for the analysis ofp63 expression in general, and in particular, as a diagnostic foranalysis of carcinomas.

Techniques for conferring immunogenicity on a protein or peptide includeconjugation to carriers or other techniques well known in the art. Animmunogenic portion of a p63 protein can be administered in the presenceof adjuvant. The progress of immunization can be monitored by detectionof antibody titers in plasma or serum. Standard ELISA or otherimmunoassays can be used with the immunogen as antigen to assess thelevels of antibodies. In a preferred embodiment, the subject antibodiesare immunospecific for antigenic determinants of a p63 protein of amammal, e.g., antigenic determinants of a protein set forth in SEQ IDNo: 2 or closely related homologs (e.g., at least 90% identical, andmore preferably at least 95% identical).

Following immunization of an animal with an antigenic preparation of ap63 polypeptide, anti-p63 antisera can be obtained and, if desired,polyclonal anti-p63 antibodies isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a mammalian p63polypeptide of the present invention and monoclonal antibodies isolatedfrom a culture comprising such hybridoma cells. In one embodimentanti-human p63 antibodies specifically react with the protein encoded bythe DNA of Hup63geno (PAC). The Hup63geno (PAC) clone was deposited withthe American Type Culture Collection (ATCC), 1081 University Blvd.,Manassas, Va. 20110, under the terms fo the Budapest Treaty. The depositwas made on Oct. 13, 1997 and received ATCC accession number 209359.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectmammalian p63 polypeptides. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example, F(ab)₂fragments can be generated by treating antibody with pepsin. Theresulting F(ab)₂ fragment can be treated to reduce disulfide bridges toproduce Fab fragments. The antibody of the present invention is furtherintended to include bispecific, single-chain, and chimeric and humanizedmolecules having affinity for a p63 protein conferred by at least oneCDR region of the antibody. In preferred embodiments, the antibodies,the antibody further comprises a label attached thereto and able to bedetected, (e.g., the label can be a radioisotope, fluorescent compound,enzyme or enzyme co-factor).

Anti-p63 antibodies can be used, e.g., to monitor p63 protein levels inan individual for determining, e.g., whether a subject has a disease orcondition associated with an aberrant p63 protein level, or allowingdetermination of the efficacy of a given treatment regimen for anindividual afflicted with such a disorder. The level of p63 polypeptidesmay be measured from cells in bodily fluid, such as in blood samples.

Another application of anti-p63 antibodies of the present invention isin the immunological screening of cDNA libraries constructed inexpression vectors such as λgt11, λgt18-23, λZAP, and λORF8. Messengerlibraries of this type, having coding sequences inserted in the correctreading frame and orientation, can produce fusion proteins. Forinstance, λgt11 will produce fusion proteins whose amino termini consistof β-galactosidase amino acid sequences and whose carboxy terminiconsist of a foreign polypeptide. Antigenic epitopes of a p63 protein,e.g., other orthologs of a particular p63 protein or other paralogs fromthe same species, can then be detected with antibodies, as, for example,reacting nitrocellulose filters lifted from infected plates withanti-p63 antibodies. Positive phage detected by this assay can then beisolated from the infected plate. Thus, the presence of p63 homologs canbe detected and cloned from other animals, as can alternate isoforms(including splicing variants) from humans.

In another embodiment, a panel of monoclonal antibodies may be used,wherein each of the epitopes involved p63 functions are represented by amonoclonal antibody. Loss or perturbation of binding of a monoclonalantibody in the panel would be indicative of a mutational attention ofthe p63 protein and thus of the p63 gene.

6. Transgenic Animals

One aspect of the present invention relates to transgenic non-humananimals having germline and/or somatic cells in which the biologicalactivity of one or more tumor supressor genes, e.g., p63, p53, p73proteins, combinations thereof, are altered by a chromosomallyincorporated transgene.

In one preferred embodiment, the transgene disrupts at least a portionof a genomic p63 gene. For instance, the transgene may delete all or aportion of the genomic p63 gene by replacement recombination, or mayfunctionally interrupt one or more of a regulatory sequence or codingsequence of the genomic p63 gene by insertion recombination.

In another preferred embodiment, the transgene encodes a p63 protein,and expression of the transgene in cells of the transgenic animalresults in altered regulation of the level of the p63 protein relativeto normal expression of the wild-type p63 protein.

In still other preferred embodiments, the transgene encodes a mutant p63protein, such as dominant negative p63 protein which antagonizes atleast a portion of the biological function of a wild-type p63 protein.

Yet another preferred transgenic animal includes a transgene encoding anantisense transcript which, when transcribed from the transgene,hybridizes with a genomic p63 gene or a mRNA transcript thereof, andinhibits expression of the genomic p63 gene.

In one embodiment, the present invention provides a desired non-humananimal or an animal (including human) cell which contains a predefined,specific and desired alteration rendering the non-human animal or animalcell predisposed to cancer. Specifically, the invention pertains to agenetically altered non-human animal (most preferably, a mouse), or acell (either non-human animal or human) in culture, that is defective inat least one of two alleles of a tumor-suppressor gene such as the p63gene. The inactivation of at least one of these tumor suppressor allelesresults in an animal with a higher susceptibility to tumor induction orother proliferative or differentiative disorders, or disorders marked byabberrant signal transduction, e/g/, from a cytokine or growth factor. Agenetically altered mouse of this type is able to serve as a usefulmodel for hereditary cancers and as a test animal for carcinogenstudies. The invention additionally pertains to the use of suchnon-human animals or animal cells, and their progeny in research andmedicine.

Furthermore, it is contemplated that cells of the transgenic animals ofthe present invention can include other transgenes, e.g., which alterthe biological activity of a second tumor suppressor gene or anoncogene. For instance, the second transgene can functionally disruptthe biological activity of a second tumor suppressor gene, such as p53,p73, DCC, p21^(cip1), p27^(kip1), Rb, Mad or E2F. Alternatively, thesecond transgene can cause overexpression or loss of regulation of anoncogene, such as ras, myc, a cdc25 phosphatase, Bcl-2, Bcl-6, atransforming growth factor, neu, int-3, polyoma virus middle T antigen,SV40 large T antigen, a papillomaviral E6 protein, a papillomaviral E7protein, CDK4, or cyclin D1.

A preferred transgenic non-human animal of the present invention hasgermline and/or somatic cells in which one or more alleles of a genomicp63 gene, a p73 gene, a p53 gene, and combinations thereof, aredisrupted by a chromosomally incorporated transgene, wherein thetransgene includes a marker sequence providing a detectable signal foridentifying the presence of the transgene in cells of the transgenicanimal, and replaces at least a portion of the genomic p63 gene or isinserted into the genomic p63 gene or disrupt expression of a wild typep63 protein.

Another aspect of the present invention relates to cells and tissuesisolated from the subject transgenic animals. For instance, the presentinvention provides composition of cells, isolated ex vivo, which includea diploid genome having a chromosomally incorporated transgene, whichtransgene functionally modifies the biological activity of one or morep63 proteins. In preferred embodiments, the transgene deletes all or aportion of a genomic p63 gene by replacement recombination, orfunctionally interrupts one or more of a regulatory sequence or codingsequence of the genomic p63 gene by insertion recombination. Forinstance, one class of such cells contemplated by the present inventioninclude transgenes which have (i) at least a portion of the genomic p63gene which directs recombination of the transgene with the genomic p63gene, and (ii) a marker sequence which provides a detectable signal foridentifying the presence of the transgene in a cell.

The animals of this invention can be used as a source of cells,differentiated or precursor, which can be immortalized in cell culture.In a preferred embodiment, the cells are stem cells or pluripotentprogenitor cells. For instance, such cells can be precursors ofhematopoietic cells, neuronal cells, pancreatic cells, hepatic cells,chondrocytes, osteocytes, myocytes, or combinations thereof.

Still another aspect of the present invention relates to methods forgenerating non-human animals and stem cells having a functionallydisrupted endogenous p63 gene. In a preferred embodiment, the methodcomprises the steps of:

-   -   (i) constructing a transgene construct including (a) a        recombination region having at least a portion of the p63 gene,        which recombination region directs recombination of the        transgene with the p63 gene, and (b) a marker sequence which        provides a detectable signal for identifying the presence of the        transgene in a cell;    -   (ii) transferring the transgene into stem cells of a non-human        animal;    -   (iii) selecting stem cells having a correctly targeted        homologous recombination between the transgene and the p63 gene;    -   (iv) transferring cells identified in step (iii) into a        non-human blastocyst and implanting the resulting chimeric        blastocyst into a non-human female; and    -   (v) collecting offspring harboring an endogenous p63 gene allele        having the correctly targeted recombination.

Yet another aspect of the invention provides a method for evaluating thecarcinogenic potential of an agent by (i) contacting a transgenic animalof the present invention with a test agent, and (ii) comparing thenumber of transformed cells in a sample from the treated animal with thenumber of transformed cells in a sample from an untreated transgenicanimal or transgenic animal treated with a control agent. The differencein the number of transformed cells in the treated animal, relative tothe number of transformed cells in the absence of treatment with acontrol agent, indicates the carcinogenic potential of the testcompound.

Another aspect of the invention provides a method of evaluating ananti-proliferative activity of a test compound. In preferredembodiments, the method includes contacting a transgenic animal of thepresent invention, or a sample of cells from such animal, with a testagent, and determining the number of transformed cells in a specimenfrom the transgenic animal or in the sample of cells. A statisticallysignificant decrease in the number of transformed cells, relative to thenumber of transformed cells in the absence of the test agent, indicatesthe test compound is a potential anti-proliferative agent.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

7. Screening Assays for p63 Therapeutics

The invention provides for p63 therapeutic compounds for treatingdiseases or conditions caused by, or contributed to by an abnormal p63activity, e.g., a predisposition to form tumors. The compounds that canbe used for this purpose can be any type of compound, including aprotein, a peptide, peptidomimetic, small molecule, and nucleic acid. Anucleic acid can be, e.g., a gene, an antisense nucleic acid, aribozyme, or a triplex molecule. A compound of the invention can be anagonist or an antagonist. Preferred p63 agonists include p63 proteins orderivatives thereof which mimic at least one p63 activity, e.g., theability to activate transcription, act as a tumor suppressor, inhibittumorigenesis by eliminating potentially tumorigeale cells hydrolysis ofa target peptide or nucleic acids encoding such. Other preferredagonists include compounds which are capable of increasing theproduction of p63 protein in cells, e.g., compounds capable ofupregulating the expression of a p63 gene. Preferred p63 antagonistsinclude compounds which decrease or inhibit interaction of a p63 proteinwith a target gene. In a preferred embodiment, a p63 antagonist is amodified form of a target peptide, which is capable of interacting withthe target gene, but which does not have biological activity, e.g., willnot act as a transcription factor.

It is possible that when p63 functions as a transcription factor, ituses its central domain to bind to its target sequence. This region ofp63 may also b a target for other proteins that interact with it. Theseproteins, could increase or decrease transactivation. Accordingly,compounds modulating the interaction of such proteins with p63 could beagonists or antagonists.

Thus, the invention provides methods for identifying p63 agonist andantagonist compounds, comprising selecting compounds which are capableof modulating the interaction of an p63 protein with another moleculereferred to herein as “p63 binding partner”. A p63 binding partner canbe a target gene or a target oncoprotein etc. A p63 binding partner canalso be a polypeptide which is not a target peptide and which may, e.g.,interact with a p63 protein at sites other than its major bindingdomain. In yet other embodiments of the invention, an p63 therapeutic isa compound which is capable of binding to a p63 protein, e.g., awild-type p63 protein or a mutated form of a p63 protein, and therebymodulate the catalytic activity of the p63-protein or degrade or causethe p63 protein to be degraded. For example, such an p63 therapeutic canbe an antibody or derivative thereof which interacts specifically withan p63 protein (either wild-type or mutated).

In a further embodiment, the p63 therapeutic of the invention is capableof acting on an p63 gene, e.g., to modulate its expression.

The compounds of the invention can be identified using various assaysdepending on the type of compound and activity of the compound that isdesired. Set forth below are at least some assays that can be used foridentifying p63 therapeutics. It is within the skill of the art todesign additional assays for identifying p63 therapeutics.

7.1. Cell-Free Assays

Cell-free assays can be used to identify compounds which modulate theinteraction between an p63 protein and a p63 binding partner, such as atarget gene or peptide. In a preferred embodiment, cell-free assays foridentifying such compounds consist essentially in combining together ina reaction mixture a p63 protein, a p63 binding partner and a testcompound or a library of test compounds. A test compound can be aderivative of a p63 binding partner, e.g., an biologically inactivetarget peptide, or the test compound can be a small molecule.

Accordingly, an exemplary screening assay of the present inventionincludes the steps of (a) forming a reaction mixture including: (i) ap63 polypeptide, (ii) a p63 binding partner (e.g., p21 such as a targetgene, examples include activation of p21, which exhibits the cell cycleand/or GADD45 a repair protein activated by pathways that respond toirradiation damage), and (iii) a test compound; and (b) detectinginteraction of the p63 and the p63 binding protein. The p63 polypeptideand p63 binding partner can be produced recombinantly, purified from asource, e.g., plasma, or chemically synthesized, as described herein. Astatistically significant change (potentiation or inhibition) in theinteraction of the p63 and p63 binding protein in the presence of thetest compound, relative to the interaction in the absence of the testcompound, indicates a potential agonist (mimetic or potentiator) orantagonist (inhibitor) of p63 bioactivity for the test compound.

The compounds of this assay can be contacted simultaneously.Alternatively, a p63 protein can first be contacted with a test compoundfor an appropriate amount of time, following which the p63 bindingpartner is added to the reaction mixture. The efficacy of the compoundcan be assessed by generating dose response curves from data obtainedusing various concentrations of the test compound. Moreover, a controlassay can also be performed to provide a baseline for comparison. In thecontrol assay, isolated and purified p63 polypeptide or binding partneris added to a composition containing the p63 binding partner or p63polypeptide, and the formation of a complex is quantitated in theabsence of the test compound.

Complex formation between a p63 protein and a p63 binding partner may bedetected by a variety of techniques. Modulation of the formation ofcomplexes can be quantitated using, for example, detectably labeledproteins such as radiolabeled, fluorescently labeled, or enzymaticallylabeled p63 proteins or p63 binding partners, by immunoassay, or bychromatographic detection.

Typically, it will be desirable to immobilize either p63 or its bindingpartner to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of p63 to a p63 binding partner, can be accomplished inany vessel suitable for containing the reactants. Examples includemicrotitre plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows the protein to be bound to a matrix. For example,glutathione-S-transferase/p63 (GST/p63) fusion proteins can be adsorbedonto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe p63 binding partner, e.g. an ³⁵S-labeled p63 binding partner, andthe test compound, and the mixture incubated under conditions conduciveto complex formation, e.g. at physiological conditions for salt and pH,though slightly more stringent conditions may be desired. Followingincubation, the beads are washed to remove any unbound label, and thematrix immobilized and radiolabel determined directly (e.g. beads placedin scintillant), or in the supernatant after the complexes aresubsequently dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level of p63protein or p63 binding partner found in the bead fraction quantitatedfrom the gel using standard electrophoretic techniques such as describedin the appended examples.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, either p63 or itscognate binding partner can be immobilized utilizing conjugation ofbiotin and streptavidin. For instance, biotinylated p63 molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive with p63can be derivatized to the wells of the plate, and p63 trapped in thewells by antibody conjugation. As above, preparations of a p63 bindingprotein and a test compound are incubated in the p63 presenting wells ofthe plate, and the amount of complex trapped in the well can bequantitated. Exemplary methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the p63binding partner, or which are reactive with p63 protein and compete withthe binding partner; as well as enzyme-linked assays which rely ondetecting an enzymatic activity associated with the binding partner,either intrinsic or extrinsic activity. In the instance of the latter,the enzyme can be chemically conjugated or provided as a fusion proteinwith the p63 binding partner. To illustrate, the p63 binding partner canbe chemically cross-linked or genetically fused with horseradishperoxidase, and the amount of polypeptide trapped in the complex can beassessed with a chromogenic substrate of the enzyme, e.g.3,3′-diamino-benzadine terahydrochloride or 4-chloro-1-napthol.Likewise, a fusion protein comprising the polypeptide andglutathione-S-transferase can be provided, and complex formationquantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein, such asanti-p63 antibodies, can be used. Alternatively, the protein to bedetected in the complex can be “epitope tagged” in the form of a fusionprotein which includes, in addition to the p63 sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharamacia, NJ).

Cell-free assays can also be used to identify compounds which interactwith a p63 protein and modulate an activity of a p63 protein.Accordingly, in one embodiment, a p63 protein is contacted with a testcompound and the actual transcription of a target gene activity of p63is monitored. In one embodiment, the ability of p63 to bind to and/or tohydrolyze a target peptide, e.g, angiotensin I or a kinin, such asbradykinin is determined. The binding affinity of p63 to a targetpeptide can be determined according to methods known in the art.Determination of the enzymatic activity of p63 can be performed with theaid of the substrate furanacryloyl-L-phenylalanyl-glycyl-glycine (FAPGG)under conditions described in Holmquist et al. (1979) Anal. Biochem.95:540 and in U.S. Pat. No. 5,259,045. The subject screening assays canbe accomplished in any vessel suitable for containing the reactants.Examples include microtitre plates, test tubes, and micro-centrifugetubes.

7.2. Cell Based Assays

In addition to cell-free assays, such as described above, the readilyavailable source of p63 proteins provided by the present invention alsofacilitates the generation of cell-based assays for identifying smallmolecule agonists/antagonists and the like. Such assays can be used,e.g., to identify compounds which modulate expression of a p63 gene,modulate translation of a p63 mRNA, or which modulate the stability of ap63 mRNA or protein. Accordingly, in one embodiment, a cell which iscapable of producing p63, [include e.g. of cells], is incubated with atest compound and the amount of p63 produced in the cell medium ismeasured and compared to that produced from a cell which has not beencontacted with the test compound. The specificity of the compound vis avis p63 can be confirmed by various control analysis, e.g., measuringthe expression of one or more control gene.

Compounds which can be tested include small molecules, proteins, andnucleic acids. In particular, this assay can be used to determine theefficacity of p63 antisense molecules or ribozymes.

In another embodiment, the effect of a test compound on transcription ofan p63 gene is determined by transfection experiments using a reportergene operatively linked to at least a portion of the promoter of an p63gene. A promoter region of a gene can be isolated, e.g., from a genomiclibrary according to methods known in the art. The reporter gene can beany gene encoding a protein which is readily quantifiable, e.g, theluciferase or CAT gene, well known in the art.

In another embodiment, the invention provides a method for detectingfunctional p63 protein in cells, preferably mammalian cells. ‘functionalp63’ means a p63 protein which is able to activate gene transcription.The invention relates to a method of determining the presence offunctional p63 based on the dependence of transactivation of certaintarget genes by p63. Examples include the p21 gene or repair proteinsfor instance those activated by irradiation damage. The method comprises(a) stimulating mammalian cells to increase expression of the targetmRNA; and (b) comparing the level of the mRNA in stimulated cells to thelevel of mRNA in unstimulated cells.

For instance, primary cultures of mammalian cells can also be used. Suchcells can be biopsies taken from mammalian tumors. Mammalian cellcultures can be initiated from biopsies by surgical incisional orescisional methods. In one embodiment, the cells may be stimulated instep (a) by irradiating the cells in order to induce or stimulateexpression of the repair proetins.

The RNA can be isolated from irradiated mammalian cells by methods knownto those skilled in the art.

7.3. Ubiquitin-Mediated Proteolysis

Furthermore, the present invention, by making available purified andrecombinant forms of the subject p63 proteins, facilitates thedevelopment of assays that can be used to screen for drugs which inhibitthe proteolysis of p63, such as by inhibiting ubiquitination of p63, orubiquitin-mediated proteolysis of p63. For instance, in addition toagents which disrupt binding of p63 to other cellular (or viral)proteins, inhibitors of ubiquitin conjugating enzymes (“E2” enzymes) orubiquitin ligases (“E3” enzymes) may prevent transfer of ubiquitin top63.

Assays for the measurement of ubiquitination can be generated in manydifferent forms, and include assays based on cell-free systems, e.g.purified proteins or cell lysates, as well as cell-based assays whichutilize intact cells. Assays as described herein can be used inconjunction with the subject p63 proteins to generate aubiquitin-conjugating system for detecting agents able to inhibitparticular E2- or E3-mediated ubiquitination of p63 proteins. Suchinhibitors can be used, for example, in the treatment of proliferativeand/or differentiative disorders, to modulate apoptosis, and in thetreatment of viral infections, such by adenoviruses or papillomaviruses.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins or with lysates, are oftenpreferred as “primary” screens in that they can be generated to permitrapid development and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with other proteinsor change in enzymatic properties of the molecular target. Accordingly,potential inhibitors of p63 ubiquitination can be detected in acell-free assay generated by constitution of a functionalubiquitin-protein ligase system in a cell lysate, such as generated bycharging a reticulocyte lysate (Hersko et al. (1983) J Biol Chem258:8206-6214) with a p63 polypeptide and, as needed, a specific E1, E2or E3 enzyme (cellular or viral in origin), and ubiquitin. In analternative format, the assay can be derived as a reconstituted proteinmixture.

In yet other embodiments, the present assay comprises an in vivoubiquitin-conjugating system, such as a cell able to conduct the p63protein through at least a portion of a ubiquitin-mediated proteolyticpathway.

The level of ubiquitination of the substrate p63 protein brought aboutby the system is measured in the presence and absence of a candidateagent, and a decrease in the level ubiquitin conjugation is indicativeof an inhibitory activity for the candidate agent. As described below,the level of ubiquitination of the p63 protein can be measured bydetermining the actual concentration of protein:ubiquitin conjugatesformed; or inferred by detecting some other quality of the subjectprotein affected by ubiquitination, including the proteolyticdegradation of the protein. A statistically significant decrease inubiquitination of the p63 protein in the presence of the test compoundis indicative of the test compound being, as appropriately inferred fromthe assay format, an inhibitor of ubiquitin conjugation to p63 and/orubiquitin-mediated degradation of p63.

In preferred in vitro embodiments of the present assay, theubiquitin-conjugating system comprises a reconstituted protein mixtureof at least semi-purified proteins. With respect to measuringubiquitination, the purified protein mixture can substantially lack anyproteolytic activity which would degrade the p63 substrate proteinand/or components of the ubiquitin conjugating system. For instance, thereconstituted system can be generated to have less than 10% of theproteolytic activity associated with a typical reticulocyte lysate, andpreferably no more than 5%, and most preferably less than 2%.Alternatively, the mixture can be generated to include, either from theonset of ubiquitination or from some point after ubiquitin conjugationof the p63 protein, a ubiquitin-dependent proteolytic activity, such asa purified proteosome complex, that is present in the mixture atmeasured amounts.

In the subject method, ubiquitin conjugating systems derived frompurified proteins hold a number of significant advantages over celllysate or wheat germ extract based assays (collectively referred tohereinafter as “lysates”). Unlike the reconstituted protein system,without knowledge of particular kinetic parameters for Ub-independentand Ub-dependent degradation of the p63 protein in the lysate,discerning between the two pathways can be extremely difficult.Measuring these parameters, if at all possible, is further made tediousby the fact that cell lysates tend to be inconsistent from batch tobatch, with potentially significant variation between preparations.Evaluation of a potential inhibitor using a lysate system is alsocomplicated in those circumstances where the lysate is charged with mRNAencoding the p63 protein, as such lysates may continue to synthesize theprotein during the assay, and will do so at unpredictable rates.

Using similar considerations, knowledge of the concentration of eachcomponent of the ubiquitin conjugation pathway can be required for eachlysate batch, along with the degradative kinetic data, in order todetermine the necessary time course and calculate the sensitivity ofexperiments performed from one lysate preparation to the next.

Furthermore, the lysate system can be unsatisfactory where the p63protein itself has a relatively short half-life, especially if due todegradative processes other than the ubiquitin-mediated pathway to whichan inhibitor is sought. For example, in assays for an inhibitor ofHPV-induced ubiquitination of p53, lysate based systems can be difficultto use, in addition to the reasons set forth above, due to the shorthalf-life of p53 even in extracts which lack HPV proteins. In suchsystems, the ability to measure HPV-mediated ubiquitination of p53 ismade difficult by the already rapid, ongoing degradation of p53presumably occurring by proteolytic processes which are not mediated byany HPV proteins.

The use of reconstituted protein mixtures allows more careful control ofthe reaction conditions in the ubiquitin-conjugating system. Moreover,the system can be derived to favor discovery of inhibitors of particularsteps of the ubiquitination process. For instance, a reconstitutedprotein assay can be generated which does not facilitate degradation ofthe ubiquitinated p63 protein. The level of ubiquitin conjugated p63 caneasily be measured directly in such as system, both in the presence andabsence of a candidate agent, thereby enhancing the ability to detect ainhibitor of p63 ubiquitination. Alternatively, the Ub-conjugatingsystem can be allowed to develop a steady state level of p63:Ubconjugates in the absence of a proteolytic activity, but then shifted toa degradative system by addition of purified Ub-dependent proteases.Such degradative systems would be amenable to identifying directinhibitors of ubiquitin-mediated proteolysis of p63.

The purified protein mixture includes a purified preparation of the p63protein and ubiquitin under conditions which drive the conjugation ofthe two molecules. For instance, the mixture can include aubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2),and a nucleotide triphosphate (e.g. ATP). Alternatively, the E1 enzyme,the ubiquitin, and the nucleotide triphosphate can be substituted in thesystem with a pre-activated ubiquitin in the form of an E1::Ubconjugate. Likewise, a pre-activated ubiquitin can instead comprise anE2::Ub conjugate which can directly transfer the pre-activated ubiquitinto the p63 protein substrate. Furthermore, the reconstituted mixture canalso be generated to include at least one auxiliary substraterecognition protein (E3) which may be, for example, of cellular or viralorigin. In illustrative embodiments described below, in order togenerate an assay which approximates the ubiquitination of p63 in HPV-16or HPV-18 infected cells, the reconstituted ubiquitin conjugating systemmay further include an E6 protein of HPV origin, as well as anE6-associated protein (E6-AP) of cellular origin.

In one embodiment of the present assay, the products of anon-degradative ubiquitin-conjugating system are separated by gelelectrophoresis, and the level of ubiquitinated p63 protein assessed,using standard electrophesis protocols, by measuring an increase inmolecular weight of the p63 protein that corresponds to the addition ofone or more ubiquitin chains. For example, one or both of the p63protein and ubiquitin can be labeled with a radioisotope such as ³⁵S,¹⁴C, or ³H, and the isotopically labeled protein bands quantified byautoradiographic techniques. Standardization of the assay samples can beaccomplished, for instance, by adding known quantities of labeledproteins which are not themselves subject to ubiquitination ordegradation under the conditions which the assay is performed.Similarly, other means of detecting electrophoretically separatedproteins can be employed to quantify the level of ubiquitination of thep63 protein, including immunoblot analysis using antibodies specific foreither the p63 protein or ubiquitin, or derivatives thereof. Asdescribed below, the antibody can be replaced with another molecule ableto bind one of either the p63 protein or ubiquitin. By way ofillustration, one embodiment of the present assay comprises the use ofbiotinylated ubiquitin in the conjugating system. The biotin label isdetected in a gel during a subsequent detection step by contacting theelectrophoretic products (or a blot thereof) with astreptavidin-conjugated label, such as a streptavidin linkedfluorochrome or enzyme, which can be readily detected by conventionaltechniques. Moreover, where a reconstituted protein mixture is used(rather than a lysate) as the conjugating system, it may be possible tosimply detect the p63 protein and ubiquitin conjugates in the gel bystandard staining protocols, including coomassie blue and silverstaining.

In another embodiment, an immunoassay or similar binding assay, is usedto detect and quantify the level of ubiquitinated p63 protein producedin the ubiquitin-conjugating system. Many different immunoassaytechniques are amenable for such use and can be employed to detect andquantitate the p63 protein:Ub conjugates. For example, the wells of amicrotitre plate (or other suitable solid phase) can be coated with anantibody which specifically binds one of either the p63 protein orubiquitin. After incubation of the ubiquitin-conjugated system with andwithout the candidate agent, the products are contacted with the matrixbound antibody, unbound material removed by washing, and ubiquitinconjugates of the p63 protein specifically detected. To illustrate, ifan antibody which binds the p63 protein is used to sequester the proteinon the matrix, then a detectable anti-ubiquitin antibody can be used toscore for the presence of ubiquitinated p63 protein on the matrix.

In similar fashion, epitope-tagged ubiquitin, such as myc-ub (seeEllison et al. (1991) J. Biol. Chem. 266:21150-21157; ubiquitin whichincludes a 10-residue sequence encoding a protein of c-myc) can be usedin conjunction with antibodies to the epitope tag. A major advantage ofusing such an epitope-tagged ubiquitin approach for detecting Ub:proteinconjugates is the ability of an N-terminal tag sequences to inhibitubiquitin-mediated proteolysis of the conjugated p63 protein.

Other ubiquitin derivatives include detectable labels which do notinterfere greatly with the conjugation of ubiquitin to the p63 protein.Such detectable labels can include fluorescently-labeled (e.g. FITC) orenzymatically-labeled ubiquitin fusion proteins. These derivatives canbe produced by chemical cross-linking, or, where the label is a protein,by generation of a fusion protein. Several labeled ubiquitin derivativesare commercially available.

Moreover, the p63 protein can be generated as aglutathione-S-transferase (GST) fusion protein. As a practical matter,such GST fusion protein can enable easy purification of the p63 proteinin the preparation of components of the ubiquitin-conjugating system(see, for example, Current Protocols in Molecular Biology, eds. Ausubelet al. (NY: John Wiley & Sons, 1991); Smith et al. (1988) Gene 67:31;and Kaelin et al. (1992) Cell 70:351) Moreover, glutathione derivatizedmatrices (e.g. glutathione-sepharose or glutathione-coated microtitreplates) can be used to sequester free and ubiquitinated forms of the p63protein from the ubiquitin-conjugating system, and the level ofubiquitin immobilized can be measured as described. Likewise, where thematrix is generated to bind ubiquitin, the level of sequestered GST-p63protein can be detected using agents which bind to the GST moiety (suchas anti-GST antibodies), or, alternatively, using agents which areenzymatically acted upon by GST to produce detectable products (e.g.1-chloro-2,4-dinitrobenzene; Habig et al. (1974) J Biol Chem 249:7130).Similarly, other fusion proteins involving the p63 protein and anenzymatic activity are contemplated by the present method. For example,fusion proteins containing β-galactosidase or luciferase, to name but afew, can be employed as labels to determine the amount of p63 proteinsequestered on a matrix by virtue of a conjugated ubiquitin chain.

Moreover, such enzymatic fusion proteins can be used to detect andquantitate ubiquitinated p63 protein in a heterogeneous assay, that isone which does not require separation of the components of theconjugating system. For example, ubiquitin conjugating systems can begenerated to have a ubiquitin-dependent protease which degrades the p63protein. The enzymatic activity of the fusion protein provides adetectable signal, in the presence of substrate, for measuring the levelof the p63 protein ubiquitination. Similarly, in a non-degradativeconjugating system, ubiquitination of the p63 protein portion of thefusion protein can allosterically influence the enzymatic activityassociated with the fusion the protein and thereby provides a means formonitoring the level of ubiquitin conjugation.

In binding assay-type detection steps set out above, the choice of whichof either the p63 protein or ubiquitin should be specificallysequestered on the matrix will depend on a number of factors, includingthe relative abundance of both components in the conjugating system. Forinstance, where the reaction conditions of the ubiquitin conjugatingsystem provide ubiquitin at a concentration far in excess of the levelof the p63 protein, (e.g., one order of magnitude or greater)sequestering the ubiquitin and detecting the amount of p63 protein boundwith the ubiquitin can provide less dynamic range to the detection stepof the present method than the converse embodiment of sequestering thep63 protein and detecting ubiquitin conjugates from the total p63protein bound to the matrix. That is, where ubiquitin is provided ingreat excess relative to the p63 protein, the percentage of ubiquitinconjugated p63 protein in the total ubiquitin bound to the matrix can besmall enough that any diminishment in ubiquitination caused by aninhibitor can be made difficult to detect by the fact that, for example,the statistical error of the system (e.g. the noise) can be asignificant portion of the measured change in concentration of bound p63protein. Furthermore, it is clear that manipulating the reactionconditions and reactant concentrations in the ubiquitin-conjugatingsystem can be carried out to provide, at the detection step, greatersensitivity by ensuring that a strong ubiquitinated protein signalexists in the absence of any inhibitor.

Furthermore, drug screening assays can be generated which do not measureubiquitination per se, but rather detect inhibitory agents on the basisof their ability to interfere with binding of p63 with any immediateupstream or downstream component of the ubiquitin conjugation orproteolysis pathways. Such assays, which are based on disruptingprotein-protein interactions, can be carried out as described above forother p63 interactors.

In still further embodiments of the present assay, theubiquitin-conjugating system is generated in whole cells, takingadvantage of cell culture techniques to support the subject assay. Forexample, as described below, the ubiquitin-conjugating system (includingthe p63 protein and detection means) can be constituted in a eukaryoticcell culture system, including mammalian and yeast cells. Advantages togenerating the subject assay in an intact cell include the ability todetect inhibitors which are functional in an environment more closelyapproximating that which therapeutic use of the inhibitor would require,including the ability of the agent to gain entry into the cell.Furthermore, certain of the in vivo embodiments of the assay, such asexamples given below, are amenable to high through-put analysis ofcandidate agents.

The components of the ubiquitin-conjugating system, including the p63protein, can be endogenous to the cell selected to support the assay.Alternatively, some or all of the components can be derived fromexogenous sources. In any case, the cell is ultimately manipulated afterincubation with a candidate inhibitor in order to facilitate detectionof ubiquitination or ubiquitin-mediated degradation of the p63 protein.As described above for assays performed in reconstituted proteinmixtures or lysate, the effectiveness of a candidate inhibitor can beassessed by measuring direct characteristics of the p63 protein, such asshifts in molecular weight by electrophoretic means or detection in abinding assay. For these embodiments, the cell will typically be lysedat the end of incubation with the candidate agent, and the lysatemanipulated in a detection step in much the same manner as might be thereconstituted protein mixture or lysate.

Indirect measurement of ubiquitination of the p63 protein can also beaccomplished by detecting a biological activity associated with the p63protein that is either attenuated by ubiquitin-conjugation or destroyedalong with the p63 protein by ubiquitin-dependent proteolytic processes.As set out above, the use of fusion proteins comprising the p63 proteinand an enzymatic activity are representative embodiments of the subjectassay in which the detection means relies on indirect measurement ofubiquitination of the p63 protein by quantitating an associatedenzymatic activity.

Where the p63 protein has a relatively short half-life due toubiquitin-dependent or independent degradation in the cell, preferredembodiments of the assay either do not require cell lysis, or,alternatively, generate a longer lived detection signal that isindependent of the p63 protein's fate after lysis of the cell. Withrespect to the latter embodiment, the detection means can comprise, forexample, a reporter gene construct which includes a positivetranscriptional regulatory element that binds and is responsive to thep63 protein. For instance, p63 responsive elements can be used toconstruct the reporter gene. These can include a creatine kinaseenhancer, an interleukin-6 promoter, a c-fos promoter, a β-actinpromoter, an hsc70 promoter, a c-jun promoter, a p53 promoter, and aCYC1 hybrid promoter containing a p53/p63-binding sequence. The geneproduct is a detectable label, such as luciferase or β-galactosidase, ora selectable marker, such as an enzyme which confers resistance toantibiotic or other drug, and is produced in the intact cell. The labelcan be measured in a subsequent lysate of the cell. However, the lysisstep is preferably avoided, and providing a step of lysing the cell tomeasure the label will typically only be employed where detection of thelabel cannot be accomplished in whole cells. Such embodiments of thesubject assay are particularly amenable to high through-put analysis inthat proliferation of the cell can provide a simple measure ofinhibition of the ubiquitin-mediated degradation of the p63 protein.

To illustrate, the plasmid pTKluc described in PCT PublicationWO95/18974 comprises a luciferase gene whose expression is driven by thecore Herpes simplex virus thymidine-kinase (TK) promoter which has beenmodified with either p53 (p53RE/TK), myc (mycRE/TK), or Sp1 (Sp1RE/TK)binding sites. This reporter gene construct is expected to be sensitiveto the level of p63 in the cell. For instance, When the constructlacking any of the modifications to the TK promoter is transfected intomammalian cells, the detectable luciferase activity should be lowbecause this core TK promoter fragment does not contain the upstreamactivating sequences necessary for efficient transcriptional activationof the luciferase gene by p53, and accordingly, should not betransactivated by p63. However transfection with the constructs in whichTK is further modified to contain either 3 or 6 response-elements (RE)for one of p53, myc or Sp1, the detectable luciferase activity shouldincreases in cells which express appropriate forms of p63. For example,the level of luciferase expression is significantly higher inp53-producing cells (e.g. ML1 cells) transfected with thep53RETK-containing construct than with the TK construct. Likewise,endogenous myc and Sp1 proteins can drive expression of the mycRE/TK andSp1RE/TK constructs. As set out above, it is expected that p63 will bedegraded by the ubiquitin pathway. However, Sp1 is not known to bedegraded by any ubiquitin-mediated pathway, and the SP1RE/TK constructcan therefore be used as a control in the present assays. Thus, in thepresence of an agent which inhibits ubiquitin-mediated degradation ofp63 in a cell harboring a p53RE/TK construct, the level of luciferaseactivity would increase relative to that in the cell not treated withthe candidate agent.

8. Diagnostic and Prognostic Assays

The present methods provide means for determining if a subject is atrisk for developing a disease or condition associated with disordermarked by an aberrant p63 activity, e.g., an aberrant level of p63protein or particular isoform thereof. As set forth below, diseases orconditions that can be caused by an abnormal p63 level or catalyticactivity include diseases or conditions caused by or contributed to byan abnormal amount of a target peptide of p63.

According to a diagnostic method of the present invention, loss of thewild-type p63 is detected. This loss may be due to either deletionaland/or point mutational events. If only a single p63 allele is mutated,an early neoplastic state is indicated. However, if both alleles aremutated then a late neoplastic state is indicated. The p63 allele whichis not deleted (i.e., that on the sister chromosome to the chromosomecarrying the deletion) can be screened for point mutations, such asmissense, and frameshift mutations. These mutations could lead tonon-functional p63 gene products. In addition, the point mutationalevents may occur in the regulatory regions, such as in the promoter ofthe p63 gene, could lead to a loss or diminution of expression of thep63 mRNA.

In order to detect the loss of the p63 wild-type gene in tissue, it ishelpful to isolate the tissue from the surrounding normal tissues. Meansfor enriching tumor preparations are known in the art, e.g., cytometry.Detection of point mutations may be accomplished by molecular cloning ofthe p63 allele (or alleles) present in tumor tissue. Alternatively, thepolymerase chain reaction can be used to amplify p63 gene sequencesdirectly from a genomic DNA preparation. The DNA sequence of theamplified sequences can be determined.

Specific deletions of the p63 gene can also be detected. For example,restriction fragment length polymorphism (RFLP) probes for the p63 genesmay also be used to score the loss of a p63 allele. Loss of thewild-type p63 genes may also be detected on the basis of the loss of thewild type expression products of p63. Such expression products includethe mRNA as well as the p63 protein product itself.

In one embodiment, the diagnostic method comprises determining whether asubject has an abnormal mRNA and/or protein level of p63, such as byNorthern blot analysis, reverse transcription—polymerase chain reaction(RT-PCR), in situ hybridization, immunoprecipitation, Western blothybridization, or immunohistochemistry. According to the method, cellsare obtained from a subject and the p63 protein or mRNA level isdetermined and compared to the level of p63 protein or mRNA level in ahealthy subject. An abnormal level of p63 polypeptide or mRNA level islikely to be indicative of an aberrant p63 activity.

In another embodiment, the diagnostic method comprises measuring atleast one activity of p63. For example, the ability to inducetransactivation of target genes, e.g. genes involved in cell cyclearrest. Comparison of the results obtained with results from similaranalysis performed on p63 proteins from healthy subjects will beindicative of whether a subject has an abnormal p63 activity.

In preferred embodiments, the methods for determining whether a subjectis at risk for developing a disease, such as a predisposition to developtumors, associated with an aberrant p63 activity is characterized ascomprising detecting, in a sample of cells from the subject, thepresence or absence of a genetic lesion characterized by at least one of(i) an alteration affecting the integrity of a gene encoding a p63polypeptide, or (ii) the mis-expression of the p63 gene. To illustrate,such genetic lesions can be detected by ascertaining the existence of atleast one of (i) a deletion of one or more nucleotides from a p63 gene,(ii) an addition of one or more nucleotides to a p63 gene, (iii) asubstitution of one or more nucleotides of a p63 gene, (iv) a grosschromosomal rearrangement of a p63 gene, (v) a gross alteration in thelevel of a messenger RNA transcript of a p63 gene, (vii) aberrantmodification of an p63 gene, such as of the methylation pattern of thegenomic DNA, (vii) the presence of a non-wild type splicing pattern of amessenger RNA transcript of a p63 gene, (viii) a non-wild type level ofa p63 polypeptide, (ix) allelic loss of a p63 gene, and/or (x)inappropriate post-translational modification of a p63 polypeptide. Asset out below, the present invention provides a large number of assaytechniques for detecting lesions in a p63 gene. These methods include,but are not limited to, methods involving sequence analysis, Southernblot hybridization, restriction enzyme site mapping, and methodsinvolving detection of absence of nucleotide pairing between the nucleicacid to be analyzed and a probe. These and other methods are furtherdescribed infra.

Specific diseases or disorders, e.g., genetic diseases or disorders, areassociated with specific allelic variants of polymorphic regions ofcertain genes, which do not necessarily encode a mutated protein. Thus,the presence of a specific allelic variant of a polymorphic region of agene in a subject can render the subject susceptible to developing aspecific disease or disorder. Polymorphic regions in genes, e.g, p63genes, can be identified, by determining the nucleotide sequence ofgenes in populations of individuals. If a polymorphic region isidentified, then the link with a specific disease can be determined bystudying specific populations of individuals, e.g, individuals whichdeveloped a specific disease, such as hypertension. A polymorphic regioncan be located in any region of a gene, e.g., exons, in coding or noncoding regions of exons, introns, and promoter region.

In an exemplary embodiment, there is provided a nucleic acid compositioncomprising a nucleic acid probe including a region of nucleotidesequence which is capable of hybridizing to a sense or antisensesequence of a p63 gene or naturally occurring mutants thereof, or 5′ or3′ flanking sequences or intronic sequences naturally associated withthe subject p63 genes or naturally occurring mutants thereof. Thenucleic acid of a cell is rendered accessible for hybridization, theprobe is contacted with the nucleic acid of the sample, and thehybridization of the probe to the sample nucleic acid is detected. Suchtechniques can be used to detect lesions or allelic variants at eitherthe genomic or mRNA level, including deletions, substitutions, etc., aswell as to determine mRNA transcript levels.

A preferred detection method is allele specific hybridization usingprobes overlapping the mutation or polymorphic site and having about 5,10, 20, 25, or 30 nucleotides around the mutation or polymorphic region.In a preferred embodiment of the invention, several probes capable ofhybridizing specifically to allelic variants are attached to a solidphase support, e.g., a “chip”. Oligonucleotides can be bound to a solidsupport by a variety of processes, including lithography. For example achip can hold up to 250,000 oligonucleotides (GeneChip, Affymetrix).Mutation detection analysis using these chips comprisingoligonucleotides, also termed “DNA probe arrays” is described e.g., inCronin et al. (1996) Human Mutation 7:244. In one embodiment, a chipcomprises all the allelic variants of at least one polymorphic region ofa gene. The solid phase support is then contacted with a test nucleicacid and hybridization to the specific probes is detected. Accordingly,the identity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment.

In certain embodiments, detection of the lesion comprises utilizing theprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligase chain reaction (LCR) (see, e.g., Landegran etal. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the p63 gene (see Abravaya et al. (1995)Nuc Acid Res 23:675-682). In a merely illustrative embodiment, themethod includes the steps of (i) collecting a sample of cells from apatient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) fromthe cells of the sample, (iii) contacting the nucleic acid sample withone or more primers which specifically hybridize to a p63 gene underconditions such that hybridization and amplification of the p63 gene (ifpresent) occurs, and (iv) detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In a preferred embodiment of the subject assay, mutations in, or allelicvariants, of a p63 gene from a sample cell are identified by alterationsin restriction enzyme cleavage patterns. For example, sample and controlDNA is isolated, amplified (optionally), digested with one or morerestriction endonucleases, and fragment length sizes are determined bygel electrophoresis. Moreover, the use of sequence specific ribozymes(see, for example, U.S. Pat. No. 5,498,531) can be used to score for thepresence of specific mutations by development or loss of a ribozymecleavage site.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the p63 gene anddetect mutations by comparing the sequence of the sample p63 with thecorresponding wild-type (control) sequence. Exemplary sequencingreactions include those based on techniques developed by Maxim andGilbert (Proc. Natl. Acad Sci USA (1977) 74:560) or Sanger (Sanger et al(1977) Proc. Nat. Acad. Sci. 74:5463). It is also contemplated that anyof a variety of automated sequencing procedures may be utilized whenperforming the subject assays (Biotechniques (1995) 19:448), includingsequencing by mass spectrometry (see, for example PCT publication WO94/16101; Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin etal. (1993) Appl Biochem Biotechnol 38:147-159). It will be evident toone skilled in the art that, for certain embodiments, the occurrence ofonly one, two or three of the nucleic acid bases need be determined inthe sequencing reaction. For instance, A-track or the like, e.g., whereonly one nucleic acid is detected, can be carried out.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labelled) RNA or DNA containing the wild-type p63sequence with potentially mutant RNA or DNA obtained from a tissuesample. The double-stranded duplexes are treated with an agent whichcleaves single-stranded regions of the duplex such as which will existdue to base pair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digest the mismatched regions.In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treatedwith hydroxylamine or osmium tetroxide and with piperidine in order todigest mismatched regions. After digestion of the mismatched regions,the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al (1988) Proc. Natl. Acad Sci USA 85:4397; Saleeba et al.(1992) Methods Enzymod. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in p63 cDNAs obtained from samplesof cells. For example, the mutY enzyme of E. coli cleaves A at G/Amismatches and the thymidine DNA glycosylase from HeLa cells cleaves Tat G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a p63 sequence,e.g., a wild-type p63 sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations or the identity of the allelic variant of apolymorphic region in p63 genes. For example, single strand conformationpolymorphism (SSCP) may be used to detect differences in electrophoreticmobility between mutant and wild type nucleic acids (Orita et al. (1989)Proc Natl. Acad. Sci. USA 86:2766, see also Cotton (1993) Mutat Res285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79).Single-stranded DNA fragments of sample and control p63 nucleic acidswill be denatured and allowed to renature. The secondary structure ofsingle-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing agent gradient to identify differences inthe mobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations or theidentity of the allelic variant of a polymorphic region include, but arenot limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation ornucleotide difference (e.g., in allelic variants) is placed centrallyand then hybridized to target DNA under conditions which permithybridization only if a perfect match is found (Saiki et al. (1986)Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci. USA 86:6230).Such allele specific oligonucleotide hybridization techniques may beused to test one mutation or polymorphic region per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations or polymorphic regions when the oligonucleotidesare attached to the hybridizing membrane and hybridized with labelledtarget DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation or polymorphic region of interest in the centerof the molecule (so that amplification depends on differentialhybridization) (Gibbs et al (1989) Nucleic Acids Res. 17:2437-2448) orat the extreme 3′ end of one primer where, under appropriate conditions,mismatch can prevent, or reduce polymerase extension (Prossner (1993)Tibtech 11:238. In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al. (1992) Mol. Cell. Probes 6: 1). It isanticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification (Barany (1991) Proc. Natl.Acad. Sci. USA 88:189). In such cases, ligation will occur only if thereis a perfect match at the 3′ end of the 5′ sequence making it possibleto detect the presence of a known mutation at a specific site by lookingfor the presence or absence of amplification

In another embodiment, identification of the allelic variant is carriedout using an oligonucleotide ligation assay (OLA), as described, e.g.,in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., Science241:1077-1080 (1988). The OLA protocol uses two oligonucleotides whichare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc.Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA.

Several techniques based on this OLA method have been developed and canbe used to detect specific allelic variants of a polymorphic region ofan SR-BI gene. For example, U.S. Pat. No. 5,593,826 discloses an OLAusing an oligonucleotide having 3′-amino group and a 5′-phosphorylatedoligonucleotide to form a conjugate having a phosphoramidate linkage. Inanother variation of OLA described in Tobe et al. ((1996) Nucleic AcidsRes 24: 3728), OLA combined with PCR permits typing of two alleles in asingle microtiter well. By marking each of the allele-specific primerswith a unique hapten, i.e. digoxigenin and fluorescein, each OLAreaction can be detected by using hapten specific antibodies that arelabeled with different enzyme reporters, alkaline phosphatase orhorseradish peroxidase. This system permits the detection of the twoalleles using a high throughput format that leads to the production oftwo different colors.

The invention further provides methods for detecting single nucleotidepolymorphisms in an p63 gene. Because single nucleotide polymorphismsconstitute sites of variation flanked by regions of invariant sequence,their analysis requires no more than the determination of the identityof the single nucleotide present at the site of variation and it isunnecessary to determine a complete gene sequence for each patient.Several methods have been developed to facilitate the analysis of suchsingle nucleotide polymorphisms.

In one embodiment, the single base polymorphism can be detected by usinga specialized exonuclease-resistant nucleotide, as disclosed, e.g., inMundy, C. R. (U.S. Pat. No. 4,656,127). According to the method, aprimer complementary to the allelic sequence immediately 3′ to thepolymorphic site is permitted to hybridize to a target molecule obtainedfrom a particular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site.Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087).As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employedthat is complementary to allelic sequences immediately 3′ to apolymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

Several primer-guided nucleotide incorporation procedures for assayingpolymorphic sites in DNA have been described (Komher, J. S. et al.,Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res.18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1993)). These methods differ from GBA TM in that they allrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. In such a format, since the signalis proportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A.-C.,et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

For mutations that produce premature termination of protein translation,the protein truncation test (PTT) offers an efficient diagnosticapproach (Roest, et. al., (1993) Hum. Mol. Genet. 2:1719-21; van derLuijt, et. al., (1994) Genomics 20:1-12). For PTT, RNA is initiallyisolated from available tissue and reverse-transcribed, and the segmentof interest is amplified by PCR. The products of reverse transcriptionPCR are then used as a template for nested PCR amplification with aprimer that contains an RNA polymerase promoter and a sequence forinitiating eukaryotic translation. After amplification of the region ofinterest, the unique motifs incorporated into the primer permitsequential in vitro transcription and translation of the PCR products.Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis oftranslation products, the appearance of truncated polypeptides signalsthe presence of a mutation that causes premature termination oftranslation. In a variation of this technique, DNA (as opposed to RNA)is used as a PCR template when the target region of interest is derivedfrom a single exon.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acid,primer set; and/or antibody reagent described herein, which may beconveniently used, e.g., in clinical settings to diagnose patientsexhibiting symptoms or family history of a disease or illness involvinga p63 polypeptide.

Any cell type or tissue may be utilized in the diagnostics describedbelow. In a preferred embodiment a bodily fluid, e.g., blood, isobtained from the subject to determine the presence of a mutation or theidentity of the allelic variant of a polymorphic region of an p63 gene.A bodily fluid, e.g, blood, can be obtained by known techniques (e.g.venipuncture). Alternatively, nucleic acid tests can be performed on drysamples (e.g. hair or skin). For prenatal diagnosis, fetal nucleic acidsamples can be obtained from maternal blood as described inInternational Patent Application No. WO91/07660 to Bianchi.Alternatively, amniocytes or chorionic villi may be obtained forperforming prenatal testing.

When using RNA or protein to determine the presence of a mutation or ofa specific allelic variant of a polymorphic region of a p63 gene, thecells or tissues that may be utilized must express the p63 gene.Preferred cells for use in these methods include megakaryocytes, whichhave been shown to express the 3 zinc finger proteins of the invention(see Examples). Alternative cells or tissues that can be used, can beidentified by determining the expression pattern of the specific p63gene in a subject, such as by Northern blot analysis.

Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, G. J., 1992, PCR in situhybridization: protocols and applications, Raven Press, NY).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles may also be assessed in such detectionschemes. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

Antibodies directed against wild type or mutant p63 polypeptides orallelic variant thereof, which are discussed above, may also be used indisease diagnostics and prognostics. Such diagnostic methods, may beused to detect abnormalities in the level of p63 polypeptide expression,or abnormalities in the structure and/or tissue, cellular, orsubcellular location of a p63 polypeptide. Structural differences mayinclude, for example, differences in the size, electronegativity, orantigenicity of the mutant p63 polypeptide relative to the normal p63polypeptide. Protein from the tissue or cell type to be analyzed mayeasily be detected or isolated using techniques which are well known toone of skill in the art, including but not limited to western blotanalysis. For a detailed explanation of methods for carrying out Westernblot analysis, see Sambrook et al, 1989, supra, at Chapter 18. Theprotein detection and isolation methods employed herein may also be suchas those described in Harlow and Lane, for example, (Harlow, E. andLane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), which is incorporatedherein by reference in its entirety.

This can be accomplished, for example, by immunofluorescence techniquesemploying a fluorescently labeled antibody (see below) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Theantibodies (or fragments thereof) useful in the present invention may,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of p63 polypeptides. Insitu detection may be accomplished by removing a histological specimenfrom a patient, and applying thereto a labeled antibody of the presentinvention. The antibody (or fragment) is preferably applied byoverlaying the labeled antibody (or fragment) onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the p63 polypeptide, but also its distribution inthe examined tissue. Using the present invention, one of ordinary skillwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection

Often a solid phase support or carrier is used as a support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

One means for labeling an anti-p63 polypeptide specific antibody is vialinkage to an enzyme and use in an enzyme immunoassay (EIA) (Voller,“The Enzyme Linked Immunosorbent Assay (ELISA)”, Diagnostic Horizons2:1-7, 1978, Microbiological Associates Quarterly Publication,Walkersville, Md.; Voller, et al., J. Clin. Pathol. 31:507-520 (1978);Butler, Meth. Enzymol. 73:482-523 (1981); Maggio, (ed.) EnzymeImmunoassay, CRC Press, Boca Raton, Fla., 1980; Ishikawa, et al., (eds.)Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,Mar., 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wavelength, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Moreover, it will be understood that any of the above methods fordetecting alterations in a gene or gene product or polymorphic variantscan be used to monitor the course of treatment or therapy.

9. Methods of Treating Diseases

In general, the invention provides methods for treating or preventing adisease caused by or contributed to by aberrant expression or activityof a p63 gene product, comprising administering to the subject aneffective amount of a pharmaceutical composition comprising a compoundwhich is capable of modulating a p63 activity, such that the disease istreated or prevented in the subject. Among the approaches which may beused to ameliorate disease symptoms involving an aberrant p63 activityare, for example, gene therapy, antisense, ribozyme, and triple helixmolecules described above. Other suitable compounds include thecompounds identified in the drug screening assays above, as well as thevarious antagonists and agonist forms of the protein.

In a preferred embodiment, the compounds of the present invention areuseful for regulating tumorigenesis. In fact, based on the significantnucleotide and amino acid sequence homology with p53, p63 is a logicaltarget for cancer therapy. The mutation spectrum of p53 provide clues tothe critical functional regions of the gene, that, when mutated,contribute to the carcinogenic process. Since about 80% of the missensemutations are in the sequence-specific DNA binding midregion of theprotein, investigators have focused on the transcription transactivatorfunction of p53. However, these missense mutations and the resultantamino acid substitutions can cause aberrant protein conformations thatalso may alter other functional domains, including those in thecarboxyl-terminus of the p53 protein. This positively charged regioncontains the putative major nuclear localization signal (amino acids316-325), the oligomerization domain (amino acids 319-360), and a DNAdamage-binding domain (amino acids 318-393). p53 sequence-specific DNAbinding and transcriptional transactivation can also be modulated bypost-translational mechanisms, including serine phosphorylation and theredox regulation of the cysteine residues responsible for binding zincto p53. The structure-function relationship revealed by the analysis ofthe p53 mutation spectrum, provides us with strategies for thedevelopment of rational cancer therapy; particularly because of theconsiderable sequence identity between p53 and p63. As discussed above,it has been observed that the sequence identity between p63 and p73alpha form is about 57.4% identity and the p63 and p73 beta form showsabout 69.7% identity at the protein level. p63 alpha form and p53exhibit 43.8% identity at the protein level.

In one embodiment, this invention is directed to the development ofdrugs to mimic the cell regulator function of p63. Strategies to screenpotential drugs are suggested by the development of assays reflectingthe biologic functions of the p63 protein: its binding to specific DNAsequences, its function as a transcription factor, its function as aninducer of apoptosis, and its ability to form complexes with cellular orviral oncoproteins. For instance, in one embodiment, apoptosis, the celldeath pathway may be enhanced by anticancer therapies. Cells exposed toagents that produce DNA damage, such as double-strand breaks, may usethe p53/p63-mediated pathway to induce apoptosis. It is known in the artthat certain cell growth factors act as survival factors of cancercells, therefore, reduction of these factors would produce apoptosis,e.g., the use of anti-EGF-recptor monoclonal antibodies, which block theEGF mediated growth signal cascade, have been shown to actsynergistically with anti-cancer drugs. Thus, modulating the survivalfactors, survival factor pathways, their cellular receptors andinhibitors provide novel methods of inhibiting tumorigenesis. It ispossible that p63 may mediate apoptosis by both transcriptionaltransactivation of genes that enhance apoptosis and transcriptiontransrepression of genes that inhibit apoptosis. These genes theirencoded proteins may also be targets for therapeutic strategies.

It is known in the art that certain DNA viruses have oncoproteins thatthat bind to p53 and inactivate its functions, it is likely that thesewould also inactivate the functions of the closely analogous p63. The E6protein of the oncogenic strains of the human papillomavirus binds top53 via E6-AP, a specific ubiquitin protein ligase and enhances thedigestion of p53, and possible the digestion of p63. Drugs that inhibiteither the formation of this protein complex or the digestion of eitherp53 or p63, might have a therapeutic benefit in tumors associated withhuman papillomavirus infections, such as cervical, penile, and rectalcarcinomas.

p63 has been mapped to chromosome 3, i.e., 3q27. Deletions of regions ofchromosome 3 have been implicated in lung, uterine, breast, testicular,and ovarian cancers, von Hippel-Lindau and 3p deletion syndrome, renalcell and nasopharyngeal carcinomas, mesotheliomas, and varioushaematological malignancies. In particular, chromosomal breaks at 3q27have been implicated in intermediate grade non-Hodgkin's lymphomas(NHL). The pathologic heterogeneity of NHL is reflected at the cellularlevel by the various pathways underlying NHL pathogenesis. Inparticular, two main categories of genetic lesions, activation ofdominantly acting oncogenes and deletion of tumor-suppression genes areknown in the art as contributing to lymphonogenesis. In particular,oncogene activation in NHL most commonly occurs through non-randomchromosomal translocations.

In many instances, these translocations involve reciprocal exchangesbetween an antigen receptor locus (the immunoglobulin loci or the TCR inB- and T-cell malignancies, respectively) and various protooncogeneloci. Once the protooncogne is juxtaposed to an antigen receptor locus,its expression becomes regulated by immunoglogulin TCR promoters andenhancers. The resulting transcriptional deregulation may be one factorcausing activation of the translocated protooncogene. Accordingly, inone embodiment it is contemplated that overexpression of the p63-B formsmay be implicated in NHL.

In yet another embodiment, the invention contemplates the use of acombination of strategies, for example, a low dose of a DNA damagingagent to arrest normal cells in G1 of the cell cycle and a delayed doseof an antimitotic agent to target the mutant p63 tumor cells thatcontinue to progress into the S phase, G₂, and mitosis.

As observed in the case of p53, it is likely that tumor derived p63mutations will target amino acid residues that are important for thestructural integrity of the core domain of p63. Failure of mutantproteins to bind to DNA has been attributed to the structural defects inthe proteins such as structural rearrangements, local unfolding of thestructure, or denaturation of the core domain. Therefore, mutant p63 canhave altered sequence specific DNA-binding and function as atranscription factor either by inhibiting its transactivator activity orby changing its specificity of DNA binding and the repertoire of genesthat are transcriptionally transactivated. Although, it would seemdifficult to reverse mutant conformations to the wild type, numerousstrategies have been considered. First, certain like p53, certain p63mutant proteins are believed to have temperature sensitive phenotypes,including increased transcription-transactivator and growth inhibitionactivities at the lower permissive temperature when compared to thenon-permissive higher temperature. Second, certain peptide drugs canlater the conformation of mutant p63 in cells. Third, certain p63mutants can still form tetramers and cooperate with transfectedwild-type p63 in the transcriptional transactivation of reporter geneconstructs. It would appear that p63 missense mutants most likely toassume a wild-type protein conformation appear to be those with asubstituted amino acid in the sequence specific DNA binding site.Mutations resulting in amino acid substitutions in the interior of thep63 protein may be a thermodynamically less stable folded structure andrequire other strategies. Tumors having these interior p63 mutationsalso bind to cellular proteins which could lead to either dominantnegative or gain of oncogenic activities. Therefore, strategies such astargeting the mutant gene by triple Dna helox and antisense approachescould result in diminishing these activities and have a therapeuticbenefit.

The efficacy of p53 gene therapy in human cancer cells has beendemonstrated in vitro by Lee et al., Cancer Metastasis Rev., 14:149-61(1995), the efficacy as xenografts in athymic nude mice has beendemonstrated by Lesoon-Wood et al., Hum. Gene Ther. 6:395-405 (1995);Clayman et al., Cancer Res. 55:1-12 (1995); and Liu et al., Cancer Res.55:3117-22 (1995). The p53 gene, i.e., at p53 complementary DNAexpression vector was successfully transferred any transfection orinfection using either a replication defective retroviral or anadenoviral vector and tumor cell growth was inhibited. In fact, thesuccess of these expression vectors has led to the approval of phase Iprotocols in humans. In yet another embodiment, the use of the p53complementary DNA expression vectors in gene therapy protocols iscontemplated.

9.1. Tumor Vaccines

The treatment of cancer with tumor vaccines has been a goal ofphysicians and scientists ever since effective immunization againstinfectious disease with vaccines was developed. In the past, major tumorantigens had not been molecularly characterized. Recent advances are,however, beginning to define potential molecular targets and strategiesand this had evolved with the principle that T-cell mediated responsesare a key target for approaches to cancer immunization. In addition,these antigens are not truly foreign and tumor antigens fit more with aself/altered self paradigm, compared to a non-self paradigm for antigensrecognized in infectious diseases. Antigens that have been used in theart include the glycolipids and glycoproteins e.g. gangliosides, thedevelopmental antigens, e.g., MAGE, tyrosinase, melan-A and gp75, andmutant oncogene products, e.g., p53, ras, and HER-2/neu. Vaccinepossibilities include purified proteins and glycolipids, peptides, cDNAexpressed in various vectors, and a range of immune adjuvants.

Yet another aspect of the present invention relates to the modificationof tumor cells, and/or the immune response to tumor cells in a patientby administering a vaccine to enhance the anti-tumor immune response ina host. The present invention provides, for examples, tumor vaccinesbased on administration of expression vectors encoding a mutant tumorsuppressor gene, e.g., p53, p73, or p63, or portions thereof, orimmunogenic preparations of polypeptides.

In general, it is noted that malignant transformation of cells iscommonly associated with phenotypic changes. Such changes can includeloss, gain, or alteration in the level of expression of certainproteins. It has been observed that in some situations the immune systemmay be capable of recognizing a tumor as foreign and, as such, mountingan immune response against the tumor (Kripke, M., Adv. Cancer Res. 34,69-75 (1981)). This hypothesis is based in part on the existence ofphenotypic differences between tumor cells and normal cells, which issupported by the identification of tumor associated antigens (TAAs)(Schreiber, H., et al. Ann. Rev. Immunol. 6, 465-483 (1988)). TAAs arethought to distinguish a transformed cell from its normal counterpart.For example, three genes encoding TAAs expressed in melanoma cells,MAGE-1, MAGE-2 and MAGE-3, have recently been cloned (van der Bruggen,P., et al. Science 254, 1643-1647 (1991)). That tumor cells undercertain circumstances can be recognized as foreign is also supported bythe existence of T cells which can recognize and respond to tumorassociated antigens presented by MHC molecules. Such TAA-specific Tlymphocytes have been demonstrated to be present in the immunerepertoire and are capable of recognizing and stimulating an immuneresponse against tumor cells when properly stimulated in vitro(Rosenberg, S. A., et al. Science 233, 1318-1321 (1986); Rosenberg, S.A. and Lotze, M. T. Ann. Rev. Immunol. 4, 681-709 (1986)). In the caseof melanoma cells both the tyrosinase gene (Brichard, V., et al. J. Exp.Med. 178:489 (1993)) and the Melan-A gene (Coulie et al. J. Exp. Med.180:35)) have been identified as genes coding for antigens recognized byautologous CTL on melanoma cells.

Induction of T lymphocytes is a critical initial step in a host's immuneresponse. Activation of T cells results in cytokine production, T cellproliferation, and generation of T cell-mediated effector functions. Tcell activation requires an antigen-specific signal, often called aprimary activation signal, which results from stimulation of aclonally-distributed T cell receptor (TcR) present on the surface of theT cell. This antigen-specific signal is usually in the form of anantigenic peptide bound either to a major histocompatibility complex(MHC) class I protein or an MHC class II protein present on the surfaceof an antigen presenting cell (APC). CD4+, helper T cells recognizepeptides associated with class II molecules which are found on a limitednumber of cell types, primarily B cells, monocytes/macrophages anddendritic cells. In most cases class II molecules present peptidesderived from proteins taken up from the extracellular environment. Incontrast, CD8+, cytotoxic T cells (CTL) recognize peptides associatedwith class I molecules. Class I molecules are found on almost all celltypes and, in most cases, present peptides derived from endogenouslysynthesized proteins. For a review see Germain, R., Nature 322, 687-691(1986).

The importance of T cells in tumor immunity has several implicationswhich are important in the development of anti-tumor vaccines. Sinceantigens are processed and presented before they are recognized by Tcells, they may be derived from any protein of the tumor cell, whetherextracellular or intracellular. In addition, the primary amino acidsequence of the antigen is more important than the three-dimensionalstructure of the antigen. Tumor vaccine strategies may use the tumorcell itself as a source of antigen, or may be designed to enhanceresponses against specific gene products. (Pardoll, D. 1993. Annals ofthe New York Academy of Sciences 690:301).

The present invention provides for various tumor vaccination methods andreagents which can be used to elicit an anti-tumor response againsttransformed cells which express/display a mutant p63, p53, or p73, orwhich have been engineered to present an antigen of a mutant p63, p53,or p73. In general, the tumor vaccine strategies of the presentinvention fall into two categorie: (1) strategies that use the tumorcell itself as a source of tumor antigen, and (2) antigen-specificvaccine strategies that are designed to generate immune responsesagainst specific antigens of mutant p63, p53, or p73s.

In general, a p63, p53, or p73 vaccine polypeptide will include at leasta portion of the p63, p53, or p73 polypeptide including a site ofmutation which, when occurring in the full-length protein, results inloss of its biological activity. Where the p63, p53, or p73 tumorvaccine comprises a sufficient portion of a mutant p63, p53, or p73protein, the p63, p53, or p73 protein can be further mutated to renderthe vaccine polypeptide biologically inactive.

In one embodiment, a tumor cell which otherwise does not express amutant p63, p53, or p73 can be rendered immunogenic as a target for CTLrecognition by association of a p63, p53, or p73 vaccine polypeptide.For example, this can be accomplished by the use of gene transfervectors. Such gene transfer vectors may be administered in anybiologically effective carrier, e.g. any formulation or compositioncapable of effectively delivering the p63, p53, or p73 vaccine gene tocells in vivo. Alternatively, cells from the patient or other hostorganism can be transfected with the tumor vaccine construct ex vivo,allowed to express the p63, p53, or p73 protein, and, preferably afterinactivation by radiation or the like, administered to an individual. Inparticular, viral vectors represent an attractive method for delivery oftumor vaccine antigens because viral proteins are expressed de novo ininfected cells, are degraded within the cytosol, and are transported tothe endoplasmic reticulum where the degraded peptide products associatewith MHC class 1 molecules before display on the cell surface (Spooneret al. (1995) Gene Therapy 2:173).

Approaches include insertion of the subject gene into viral vectorsincluding recombinant retroviruses, adenovirus, adeno-associated virus,vaccinia virus, and herpes simplex virus-1, or plasmids. Viral vectorstransfect cells directly; plasmid DNA can be delivered with the help of,for example, cationic liposomes (lipofectin) or derivatized (e.g.antibody conjugated), polylysine conjugates, gramacidin S, artificialviral envelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO₄ precipitation carried out invivo. It will be appreciated that because transduction of appropriatetarget cells represents the critical first step in gene transfer, choiceof the particular gene delivery system will depend on such factors asthe phenotype of the intended target and the route of administration,e.g. locally or systemically.

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a subjectp63, p53, or p73 polypeptide in the tissue of an animal in order toellicit a cellular immune response. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the vaccine gene by thetargeted cell. Exemplary gene delivery systems of this type includeliposomal derived systems, poly-lysine conjugates, and artificial viralenvelopes.

In another embodiment the mutant p63, p53, or p73 peptides of thepresent invention may be directly delivered to the patient. Althoughsuch expression constructs as exemplified above have been shown to be anefficient means by which to obtain expression of peptides in the contextof class I molecules, vaccination with isolated peptides has also beenshown to result in class I expression of the peptides in some cases. Forexample, the use of synthetic peptide fragments containing CTL epitopeswhich are presented by class I molecules has been shown to be aneffective vaccine against infection with lymphocytic choriomeningitisvirus (Schultz et al. 1991. Proc. Natl. Acad. Sci. USA 88:2283) orsendai virus (Kast et al. 1991. Proc Natl Acad. Sci. 88:2283).Subcutaneous administration of a CTL epitope has also been found torender mice resistant to challenge with human papillomavirus16-transformed tumor cells (Feltkamp et al. (1993) Eur. J. Immunol.23:2242-2249). It is contemplated that such peptides may be presented inthe context of tumor cell class I antigens or by other, host-derivedclass I bearing cells (Huang et al. 1994. Science 264:961).

The mutant p63, p53, or p73 proteins, and portions thereof, may be usedin the preparation of vaccines prepared by known techniques (c.f., U.S.Pat. Nos. 4,565,697; 4,528,217 and 4,575,495). p63, p53, or p73polypeptides displaying antigenic regions capable of elicitingprotective immune response are selected and incorporated in anappropriate carrier. Alternatively, an antitumor antigenic portion of ap63, p53, or p73 protein may be incorporated into a larger protein byexpression of fused proteins.

The p63, p53, or p73 antitumor vaccines above may be administered in anyconventional manner, including oranasally, subcutaneously,intraperitoneally or intramuscularly. The vaccine may further comprise,as discussed infra, an adjuvant in order to increase the immunogenicityof the vaccine preparation.

In some cases it may be advantageous to couple the p63, p53, or p73polypeptide vaccine to a carrier, in particular a macromolecularcarrier. The carrier can be a polymer to which the p63, p53, or p73polypeptide is bound by hydrophobic non-covalent interaction, such as aplastic, e.g., polystyrene, or a polymer to which the polypeptide iscovalently bound, such as a polysaccharide, or a polypeptide, e.g.,bovine serum albumin, ovalbumin or keyhole limpet hemocyanin. Thecarrier should preferably be non-toxic and non-allergenic. The p63, p53,or p73 polypeptide may be multivalently coupled to the macromolecularcarrier as this provides an increased immunogenicity of the vaccinepreparation. It is also contemplated that the p63, p53, or p73polypeptide may be presented in multivalent form by polymerizing thepolypeptide with itself.

In addition, the vaccine formulations may also contain one or morestabilizer, exemplary being carbohydrates such as sorbitol, mannitol,starch, sucrose, dextrin, and glucose, proteins such as albumin orcasein, and buffers such as alkaline metal phosphate and the like.

The inclusion of CD4+ epitopes in the tumor vaccine in order to furtherenhance an anti-tumor response is also within the scope of theinvention.

In other embodiments, the tumor cell itself can be used as the source ofantitumor p63, p53, or p73 antigens. See, for review, Pardoll, D. 1993.Annals of the New York Academy of Sciences 690:301. For example, cellswhich have been identified through phenotyping as expressing a mutantp63, p53, or p73 can be used to generate a CTL response against a tumor.For example, tumor-infiltrating lymphocytes (TILs) may be derived fromtumor biopsies which have such a phenotype. Following such protocols asdescribed by Hom et al. (1991) J Immunotherap 10:153, TILs can beisolated from tumor specimens and grown in the presence of interleukin-2in order to generate oligoclonal populations of activated T-lymphocytesthat are cytolytic to the tumor cells expressing the mutant p63, p53, orp73.

In other embodiments, whole cell vaccines can be used to treat cancerpatients. Such vaccines can include, for example, irradiated autologousor allogenic tumor cells which express (endogenously or recumbently) amutant p63, p53, or p73 polypeptide (or fragment thereof), or lysates ofsuch cells.

In clinical settings, the therapeutic compound of the present inventioncan be introduced into a patient by any of a number of methods, each ofwhich is familiar in the art. For instance, a pharmaceutical preparationof the gene delivery system or peptide can be introduced systemically,e.g. by intravenous injection, and specific transduction of the proteinin the target cells occurs predominantly from specificity oftransfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle or peptide can be introduced bycatheter (see U.S. Pat. No. 5,328,470) or by stereotactic injection(e.g. Chen et al. (1994) PNAS 91: 3054-3057). A vaccine gene can bedelivered in a gene therapy construct by electroporation usingtechniques described, for example, by Dev et al. ((1994) Cancer TreatRev 20:105-115).

The pharmaceutical preparation of the vaccine therapy construct orpeptide can consist essentially of the gene delivery system in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery system can be produced intact from recombinant cells, e.g.retroviral or adenoviral vectors, the pharmaceutical preparation cancomprise one or more cells which produce the gene delivery system.

Suitable pharmaceutical vehicles for administration to a patient areknown to those skilled in the art. For parenteral administration, thep63, p53, or p73 immunogen will usually be dissolved or suspended insterile water or saline. For enteral administration, the immunogen willbe incorporated into an inert carrier in tablet, liquid, or capsularform. The preparation may also be emulsified or the active ingredientencapsulated in liposome vehicles. The composition or formulation to beadministered will, in any event, contain a quantity of the p63, p53, orp73 polypeptide adequate to achieve the desired immunized state in thesubject being treated. The immunogen preparations according to theinvention may also contain other peptides or other immunogens.

Suitable carriers may be starches or sugars and include lubricants,flavorings, binders, and other materials of the same nature. Forinstance, the immunogen can be formulated as a pharmaceuticallyacceptable acid- or base-addition salt, formed by reaction withinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid,and organic acids such as formic acid, acetic acid, propionic acid,glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid,succinic acid, maleic acid, and fumaric acid, or by reaction with aninorganic base such as sodium hydroxide, ammonium hydroxide, potassiumhydroxide, and organic bases such as mono-, di-, trialkyl and arylamines and substituted ethanolamines.

The immunogen, which may be coupled to a carrier, is preferablyadministered after being mixed with immunization adjuvants. Conventionaladjuvants include, for example, complete or incomplete Freund'sadjuvant, aluminum hydroxide, Quil A, EMA, DDA, TDM-Squalene, lecithin,alum, saponin, and such other adjuvants as are well known to those inthe art, and also mixtures thereof. For example, the p63, p53, or p73immunogen may be mixed with the N-butyl ester (murabutide) of themuramyl dipeptide (MDP;N-acetyl-glucosamine-3-yl-acetyl-L-alanyl-D-isoglutamine) diluted in asaline solution. The mixture may then be emulsified by means of an equalvolume of squalene in the presence of arlacel (excipients). It is alsopossible to use other adjuvants such as analogues of MDP, bacterialfractions such as streptococcal preparations (OK 432), Biostim (01K2) ormodified lipopolysaccharide preparations (LPS), peptidoglycans (N-Opaca)or proteoglycans (K-Pneumonia). In the case of these excipients,water-in-oil emulsions are preferable to oil-in-water emulsions.

The use of the instant invention is predicted to be of benefit in thetreatment of any type of tumor which harbors a mutant p63, p53, or p734gene. For example, treatment of tumors of the lung, breast, brain, bone,skin, bladder, kidney, ovary, or lymphocytes is contemplated. In apreferred embodiment the tumor vaccine of the present invention is usedto treat melanoma.

In addition to enhancing the immune response against a tumor at itsoriginal site, the tumor cell vaccine of the current invention may alsobe used in a method for preventing or treating metastatic spread of atumor or preventing or treating recurrence of a tumor. Thus,administration of modified tumor cells or modification of tumor cells invivo as described herein can provide tumor immunity against cells of theoriginal, unmodified tumor as well as metastases of the original tumoror possible regrowth of the original tumor.

Subjects develop an anti-tumor specific T cell response which isspecific for mutant forms of p63, p53, or p73 proteins and is effectivein keeping the patients disease free. Thus, the subject developsanti-tumor specific immunity. It is also contemplated that the inventionmay be useful in inducing immunity to tumors in susceptible individualsbefore they arise, for example in the case of familial malignancies. Astrong hereditary component has been identified for certain types ofmalignancies, for example certain breast and colon cancers and insusceptability to melanoma. In families with a known susceptibility to aparticular malignancy and in which one individual presently has a tumorbearing a mutant p63, p53, or p73 protein, peptides presented by class Imolecules of these patients could be administered to susceptible,histocompatible family members to induce an anti-tumor response in therecipient against the type of tumor to which the family is susceptible.This anti-tumor response could provide protective immunity to subsequentdevelopment of a tumor in the immunized recipient.

9.1. Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining The Ld50 (The Dose Lethal To 50% Of ThePopulation) And The Ed50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic induces are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

9.2. Formulation and Use

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For such therapy, the compounds of the invention can be formulated for avariety of loads of administration, including systemic and topical orlocalized administration. Techniques and formulations generally may befound in Remington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. For systemic administration, injection is preferred,including intramuscular, intravenous, intraperitoneal, and subcutaneous.For injection, the compounds of the invention can be formulated inliquid solutions, preferably in physiologically compatible buffers suchas Hank's solution or Ringer's solution. In addition, the compounds maybe formulated in solid form and redissolved or suspended immediatelyprior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives. in addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the oligomers of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing.

In clinical settings, a gene delivery system for the therapeutic p63gene can be introduced into a patient by any of a number of methods,each of which is familiar in the art. For instance, a pharmaceuticalpreparation of the gene delivery system can be introduced systemically,e.g., by intravenous injection, and specific transduction of the proteinin the target cells occurs predominantly from specificity oftransfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen et al.(1994) PNAS 91: 3054-3057). A p63 gene, such as any one of the sequencesrepresented in the group consisting of SEQ ID NOS 1 and 3 or a sequencehomologous thereto can be delivered in a gene therapy construct byelectroporation using techniques described, for example, by Dev et al.((1994) Cancer Treat Rev 20:105-115).

The pharmaceutical preparation of the gene therapy construct or compoundof the invention can consist essentially of the gene delivery system inan acceptable diluent, or can comprise a slow release matrix in whichthe gene delivery vehicle or compound is imbedded. Alternatively, wherethe complete gene delivery system can be produced intact fromrecombinant cells, e.g., retroviral vectors, the pharmaceuticalpreparation can comprise one or more cells which produce the genedelivery system.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

9.3. Kits

The invention further provides kits for use in diagnostics or prognosticmethods or for treating a disease or condition associated with anaberrant p63 protein. In one embodiment, the kit comprises apharmaceutical composition containing an effective amount of an p63antagonist therapeutic and instructions for use in treating orpreventing tumorigenesis. In yet another embodiment, the kit comprises apharmaceutical composition comprising an effective amount of an p63agonist therapeutic.

Yet other kits can be used to determine whether a subject has or islikely to develop a disease or condition associated with an aberrant p63activity. Such a kit can comprise, e.g., one or more nucleic acid probescapable of hybridizing specifically to at least a portion of an p63 geneor allelic variant thereof, or mutated form thereof.

10. Additional Uses for p62 Proteins and Nucleic Acids

The p63 nucleic acids of the invention can further be used in thefollowing assays. The p63 gene can also be used as a chromosomal markerin genetic linkage studies involving genes other than p63.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references (including literature references, issued patents,published patent applications as cited throughout this application arehereby expressly incorporated by reference. The practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual,2^(nd) Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames &S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, AlanR. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986);B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986). All references citedherein are incorporated by reference in their entirety.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Identification of Human and Murine p63

It has been observed that the intron-exon organization was conservedbetween p73 and p53 (Kaghad et al., 1997), and from known exon andintron sizes for these two genes, it was possible to identify newmembers of this gene family using a PCR-based strategy of amplifying twoexons in a conserved domain and their intervening intron. Sequencesimilarity in exonic regions would demonstrate a related gene, whiledifferences in size and/or sequence or introns from p53 and p73 wouldindicate a novel family member. Non-degenerate and degenerate primerswere designed based in sequence homology among p53 and p73 cDNAs fromvarious species. Primers (5′-GGCCTCGAGTACAAITWCATGTGTAAYAG (SEQ ID No:27) and 5′GGCATCGATTCTCTTCCAGGGCAAGCACA (SEQ ID No: 28)), designed toanneal to regions in exon 7 and exon 8, respectively, of p73 and p53,were used to amplify products from human and mouse total genomic DNAwith the following conditions:

-   -   Pre-PCR: 80° C. 2 min, add TAQ polymerase, 94° C. 5 min.    -   ‘Touchdown PCR’: 94° C. 1 min, 65° C. 1 min, 72° C. 2 min for 3        cycles; 94° 1 min, 64° C. 1 min, 72° C. 2 min for 3 cycles;        94° C. 1 min, 63° C. 1 min, 72° C. 2 min for 3 cycles; 94° C. 1        min, 62° C. 1 min, 72° C. 2 min for 2 cycles; 94° C. 1 min,        61° C. 1 min, 72° C. 2 min for 2 cycles; 94° C. 1 min, 60° C. 1        min, 72° C. 2 min for 2 cycles; 94° C. 1 min, 59° C. 1 min,        72° C. 2 min for 2 cycles; 94° C. 1 min, 58° C. 1 min, 72° C.2        min for 20 cycles; 72° C. 7 min.

The above generated amplicons of approximately 800 bp and 900 bp fromhuman and mouse genomic DNA, respectively. Comparison with known sizesof the corresponding intron (7) in p73 and p53 indicated that theseamplicons were derived from a novel gene that shared homology in exonicregions, as demonstrated by their ability to be generated with the aboveprimers. The amplicons were then subcloned into a pcDNA3 or pcDNA3 orpcDNA3-GFP vector and sequenced using T7, SP6, and GFP primers. Sequenceanalysis confirmed homology with regions in exon 7 and exon 8 of the p73and p53 genes, showing strong nucleotide similarity and near identity atthe amino acid level, particularly to p73.

Example 2 Cloning of p63 Via Exon-Bridging

The recent discovery of the p53-related gene, p73, suggested that othermembers of the p53 gene family exist in mammalian genomes. Given theabsence of p53-related sequences in available expressed sequence tag(EST) libraries, it was possible that p53-related genes were expressedat relatively low levels or in a tissue-restricted manner, and thatstandard hybridization or polymerase chain reaction (PCR) approacheswould be difficult. However, examination of the central portions of p53and p73 showed a remarkable conservation of intron positions, while thesize and sequences of these introns remained distinct between thesegenes (Kaghad et al., 1997; Yang et al., submitted). We thereforedesigned PCR primers that would anneal to regions in contiguous exonsconserved between p53 and p73 such that the amplification product wouldinclude the intervening intron. As products from the p53 and p73 geneswere predictable on the basis of known intron sizes and sequence, weanalyzed novel products for ones possessing terminal sequence homologywith p53 and p73 bordering non-conserved intronic sequences. One primerpair designed to span intron 7 yielded an 800 basepair (bp) and 900 bpPCR product from human and mouse, respectively, genomic DNA (not shown).The ends of these products displayed sequence homology with the 3′ endof exon 7 and the 5′-end of exon 8 of p53 and p73, while the interveningsequence showed no homology with introns of either gene. The 800 bpamplicon derived from human genomic DNA was used as a hybridizationprobe to screen a human PAC library, which yielded a single clone of 120Kb. Similarly, the 900 bp amplicon from the mouse genomic DNA was usedto isolate a single, 16.5 Kb clone from a lambda DASH II murine genomicDNA phage library. Partial sequencing of exon 7 of the human and mousegenomic clones confirmed the presence of exonic sequences withsimilarity to those of p53 and p73, and yet the presence of third basecodon differences and of conserved substitutions, demonstrating thatthis gene was a novel member of the p53 family.

To deduce the amino acid sequence of the protein, or proteins, encodedby this gene, we constructed a cDNA library from E15 murine embryoslacking p73 and p53 genes (p73−/−, p53−/−; Yang et al., unpublishedresults) and screened pools of the fractionated library by PCR andsubsequent hybridization. Three full-length cDNAs were obtained, all ofwhich shared a central domain with approximately 60 and 85% amino acididentity with the DNA binding domains of p53 and p73, respectively (FIG.1). Human cDNAs were deduced from sequencing reversetranscriptase-polymerase chain reaction (RT-PCR) products from theSK-N-MC neuroepithelioma cell line, and these revealed a remarkably highlevel of primary amino acid sequence conservation, bordering on 99%,with the mouse cDNAs (FIG. 1). Of the three murine cDNAs initiallyisolated, one encoded an acidic N-terminus similar to that required fortranscriptional activation by p53 and p73. Interestingly, the murinecDNA clone with the acidic N-terminus contains an additional 39 aminoacids upstream of the methionine start site seen in human sequences todate (FIG. 1, 2). These N-termini have been denoted TA* for the longerN-terminus and TA for the sequence starting with the amino acid sequenceMSQ (FIG. 1, 2). The other two murine cDNAs clones encoded proteins witha truncated N-terminus (ΔN) lacking the acidic, putative transactivationdomain. Further transcript and cDNA analysis from both murine and humansources revealed the expression of additional variants. In total, atleast six different transcripts, derived from alternative splicingevents and encoding proteins with two different N-termini (TA*/TA andΔN) and three different C-termini (α, β, and γ), are described (FIGS.2A-D). Partial analysis of the murine and human genes indicated that thetranscripts that give rise to the truncated N-terminal proteins werederived from an alternative promoter and initiation codon in intron 3(exon 3′; FIG. 2A). Additionally, a splicing variant in exon 9 of bothspecies alternatively deleting four amino acids was detected in bothspecies (not shown). To reflect the high degree of homology of the humanand murine sequences to p53 and p73, as well as the immense complexityof these gene products with predicted molecular weights ranging from44,000 to 72,000 daltons, we propose that this gene be called p63.

Murine ΔNp63α, ΔNp63β, TA*p63α and TA*p63β share a common core domain(SEQ ID NO: 51 (nucleic acid) and SEQ ID NO: 52 (amino acid)). MurineΔNp63γ and TA*p63γ share a common core domain (SEQ ID NO: 53 (nucleicacid) and SEQ ID NO: 54 (amino acid)). The six human p63 isoforms sharea common core domain (SEQ ID NO: 55 (nucleic acid) and SEQ ID NO: 56(amino acid)).

To map the human p63 gene, we employed fluorescence in situhybridization (FISH) techniques on human metaphase chromosome spreadsusing the human p63 PAC clone as a probe. The p63 PAC clone hybridizedto the long arm of chromosome 3, at approximately 3q27-29 (FIG. 3A). Wealso mapped the murine p63 gene using linkage analysis, which showedthat p63 is located on the proximal portion of chromosome 16 betweenanonymous DNA markers D 16Mit 1 and D16Mit3 (FIG. 3B). This region isknown to be syntenic with human chromosome 3q27-29, in agreement withthe in situ analysis of the human p63 gene.

Hup63geno (PAC) has since been deposited at the ATCC (10801 UniversityBlvd., Masassas, Va. 20110) under the terms of the Budapest Treaty. Thedeposit was made on Oct. 13, 1997 and received ATCC accession number209359.

Example 3 Mapping of Human p63 Gene

To map the human p63 gene, we used fluorescence in situ hybridization(FISH) on human metaphase chromosome spreads using the human p63 PACclone as a probe. The p63 PAC clone hybridized to the long arm ofchromosome 3 at approximately 3q27-28 (FIG. 2A). Fluorescence in situhybridization (FISH) was performed on human metaphase spreads usingHupo63geno (PAC) as a probe. The methods used protocols that are wellknown in the art. By this method, p63 was mapped to human chromosome3q27-29, a region implicated in various human syndromes including B-celllymphoma and large diffuse cell lymphoma.

Example 4 Cloning of Human p63 cDNAs

Sequence information for human p63 transcripts were obtained by RT-PCRon total RNA isolated from the SK-B-MC cell line.

Example 5 Cloning of Murine p63 Gene

The 900 bp amplicon derived from mouse genomic DNA, described above, wasused as a probe in hybridization screens for the murine homolog of thep63 gene. Screening of a 129 mouse genomic phage library (in lambda DASHII) yielded a clone with a 16.5 kb insert containing the murine p63gene. Hybridization screens were done as per standard protocols, underthe following conditions: prehybridization incubation (without probe)for 4 hrs at 50-55° C., in hybridization solution (50% formamide, 5×SSC,2.5×Denhardts, 150 ug/ml salmon sperm DNA, 0.1% SDS); hybridization with³²P-labeled probe (in hybridization solution) for 16 hrs at 40° C.;washes done in 0.5×SSC, 0.1% SDS at 50° C. The 16.5 kb insert wasreleased by a NotI digestion, subcloned into the pZero vector, andsequenced in its entirety. Sequence analysis showed the clone to containa portion of the murine p63 gene, extending from intron 4 through intron10.

Example 6 Cloning of Murine p63 cDNAs

5′ Rapid Amplification of cDNA Ends (RACE) was used to obtain furthersequence information on p63 not contained within the murine genomicclone. Total RNA was isolated from c15 embryos lacking both p53 and p73,generated from mice bearing targeted mutations in both genes, and usedas the template in a first stand cDNA synthesis reaction with a murinep63-specific primer (5′-GGCATCGATGAACTCACGGCTCAGCTC (SEQ ID No: 29)). An‘adapter’ primer (5′-TTTAGTGAGGGTTAATAAGCGGCCGCGTCGTGACTGGGAGCGC (SEQ IDNo: 30)) was then ligated to the cDNA product using T4 RNA ligase. PCRwas subsequently performed on the ligation product using primers(5′-GCCCTGGAGGCGGCCGCTTATTAACCCTCAC (SEQ ID No: 31) and5′-GGCATCGATGTAGACAGGCATGGCACG (SEQ ID No: 32)) with the conditionsdescribed in I. An approximately 610 bp amplicon was generated,subcloned into pcDNA3 vector, and sequenced in its entirety. The 5′RACEproduct yielded a sequence corresponding to a N-terminal truncated formof murine p63.

A bacterial plasmid cDNA library was constructed from mRNA isolated frome15 embryos lacking both p53 and p73, described above, and screened forp63 cDNAs. Hybridization screens were done essentially as described inV., using a probe, corresponding to exons 5 to 9 of p63, generated byRT-PCR on total p73−/−;p53−/− mouse RNA with primers(5′-GGGCTCGAGCTGAAGAAGCTGTACTGC (SEQ ID No: 33) and5′-GGGATCGATCTCCGTTTCTTGATGGAA (SEQ ID No: 34)). Three clones wereidentified and sequenced in their entirety. These corresponded to threedifferent, full-length splice variants of murine p63.

Example 7 Murine p63 Gene Targeting Vector

The 16.5 kb genomic fragment described in V. was used to construct avector for targeted disruption of the p63 gene by homologousrecombination in murine embryonic stem (ES) cells. Briefly, the p63 genefragment was subcloned into the pBluescript SK(−) vector via a NotI/NarIdigestion followed by ligation to compatible cohesive ends generated bya NotI/ClaI digestion of the vector plasmid. This construct,pSK-murp63geno, was then digested with SpeI and ClaI to remove a 2900 bpregion corresponding to portions of intron 5, all of exon 6, intron 6,exon 7, intron 7, exon 8, and portions of intron 9 (note: exon andintron designations based on exon sequence homology with p73 and p53).This region was then replaced with the neomycin resistance gene undercontrol of the PGK promoter (PGK-NeoR), yielding pSK-murp63genoNeo. Thisplasmid was then digested with SacII/NheI, removing a 2200 bp fragmentcorresponding to a portion of intron 4 that was then replaced with theherpes simplex virus thymidine kinase gene (HSV-TK), yielding the finalp63 gene targeting vector, pSK-murp63ko. This vector will be linearizedand introduced into murine ES cells via electroporation. Doubleselection with G418 and gancyclovir, followed by DNA hybridization withexternal probes will identify ES clones which have under gone homologousrecombination. These will then be used in blastocyst reconstitutions togenerate chimeric mice bearing the targeted disruption in p63. Breedingof these chimeric mice with wildtype mice and intercrosses fromsubsequent F1 and F2 generations will yield mice deficient in one orboth copies of the p63 gene.

Example 8 Cloning of Human p63 and Possible Novel, Related Genes UsingMurine p63 cDNA

We obtained a human genomic library enriched for chromosome 22 from theATCC and probed with a murine p63 cDNA fragment corresponding to exons 5to 9, described in VI. Hybridizations were done as described in V, butlower stringency washes were used (5×SSC/0.1% SDS). These screensyielded one clone containing a 4 kb insert that was then subcloned intopZero vector and sequenced in its entirety. Sequence analysis showedthat this clone was identical to previously obtained human p63 cDNAs inthe corresponding exonic regions. This result demonstrated the abilityto clone the human p63 gene using a cross-species (mouse) cDNA probe.Additional clones which yielded positive signals in our hybridizationscreens have been identified and will be purified and sequenced todetermine if they are novel members of the p73, p63, p53 family.

Example 9 Immunofluorescence

Transfection of baby hamster kidney (BHK) cells with myc-epitope taggedp63 cDNAs in pcDNAA3 vector and subsequent immunofluorescence detectionof protein was done essentially as described in Heald et al., 1993.

Example 10 Expression of p63 in Human and Murine Tissues

As the p63 cDNAs were derived from murine embryos and human cell lines,it was important to determine their expression in normal adult tissues.To address this issue, we immunized mice with bacterially-expressedglutathione-S-transferase-p63 fusion proteins, and produced an array ofmonoclonal antibodies which recognize an epitope common to murine andhuman p63 and ΔN-p63 proteins (Experimental Procedures). Using one ofthese antibodies, the 4A4 clone, we probed paraffin sections of archivalnormal human tissues including foreskin, cervix, vaginal epithelium,urothelium, and prostate (FIG. 4A-D). In all of these tissues, the 4A4monoclonal antibody showed strong nuclear staining concentrated in thebasal cells of the epithelium. The predominant localization of p63 tothe basal layer of these stratified squamous and transitional epitheliais interesting in that these cells act as the progenitors of thesuprabasal cells, which undergo differentiation and cell death inregenerative epithelia (Jetten and Harvat, 1997). In the prostate, therelationship between these p63-positive basal cells and the luminalcells is less well established, but it is thought that the basal cellsare likewise the progenitors of the suprabasal, secretory cells (Myersand Grizzle, 1997).

To obtain a more extensive survey of p63 expression in adult tissues, weprepared total RNA from various murine tissues and performed RT-PCRreactions specific for the two different p63 N-termini, TA and ΔN. Thisanalysis revealed the presence of transcripts encoding TAp63 variants inheart, testis, kidney/adrenal, thymus, brain (minus cerebellum), andcerebellum (FIG. 5 a). The ΔNp63 transcript was detected in thekidney/adrenal, spleen, and thymus, but absent from the heart, liver,testis, and brain, despite the normalization of template RNAconcentration in each reaction (FIG. 5B,C). This RT-PCR analysisindicated that TAp63 and ΔNp63 transcripts are widely expressed in adulttissues.

The immunohistochemistry on human epithelial tissues revealed highlevels of p63 expression in basal cells (FIG. 3). To determine which p63isotypes were expressed in these cells, RT-PCR reactions were performedon RNA prepared from primary human foreskin keratinocytes, humanectocervix, and ME180 cervical carcinoma cells. While RNA derived fromthe ME180 cells yielded a positive signal in the RT-PCR reactiondesigned to amplify transcripts encoding the acidic N-terminus, theprimary keratinocytes and the ectocervical cells showed little or noproduct (FIG. 5D, TA). In contrast, RNA from all three sources showedrobust signals in the RT-PCR reaction designed to amplify the ΔNp63transcript (FIG. 5D, ΔN).

The analysis of RNA from primary keratinocytes indicated that the majorp63 transcripts in these cells encoded ΔNp63 isotypes. To address thispossibility at the protein level, we performed immunoblotting on proteinlysates derived from human primary foreskin keratinocytes (HFK) andME180 cervical carcinoma cells with the 4A4 monoclonal antibody. Lysatesof baby hamster kidney (BHK) cells transfected with mammalian expressionvectors encoding epitope-tagged p63 isotypes were included as controlsfor molecular weight comparison (FIG. 5E). Significantly, the majorproduct detected in primary keratinocytes and ME180 migrates atapproximately 8okDa, slightly faster than the Myc epitope-tagged ΔNp63α.(FIG. 5E). The ME180 cells also express a less abundant, thoughdetectable, product migrating at approximately 90-95 kDa, similar tothat of TAp63α (FIG. 5E). These data, taken together with the abundantΔNp63 RT-PCR product from the ME180 cell line and primary keratinocyteRNA, are consistent with notion that these epithelial cellspredominantly express the ΔNp63α isotype.

Example 11 Transactivation Functions of p63 Isotypes

The central domain of all p63 variants is highly homologous with the DNAbinding domains of p53 and p73, suggesting that at least some p63isotypes function as transcriptional activators. The ΔNp63 variants,however, lack the N-terminal acidic residues thought to participate intransactivation functions of p53 and p73 (Ko and Prives, 1996; Levine,1997; Kaghad et al., 1997). To determine whether any of the p63 isotypescan act as transactivators, we tested their ability to induce expressionfrom a reporter gene under the control of a p53-responsive element. Sixp63 constructs, specifically TAp63α, TAp63γ, ΔNp63α, ΔNp63γ, TA*p63α,and TA*p63γ, as well as wildtype and mutant p53 expression vectors, wereseparately expressed in Saos-2 human osteosarcoma cells, which lackendogenous p53, along with a β-galactosidase reporter constructcontaining multiple copies of a minimal p53 binding sequence (PG-13, Kemet al., 1992). Lysates from cells expressing wildtype p53 yielded astrong β-galactosidase signal, while those expressing the p53 mutantshowed only a background signal (FIG. 6). Of the p63 isotypes tested inSaos-2 cells, only TAp63γ exhibited strong transcriptional activation ofthe p53 reporter, with levels approaching 80% of that seen with p53.Interestingly, the TA*p63γ isotype, which has a 39 amino acid N-terminalextension not found in TAp63γ, proved to be a weak transactivator inSaos-2 cells, suggesting possible regulatory elements within thisadditional domain. As expected, neither the ΔNp63γ nor the ΔNp63αvariant, both of which lack the putative transactivation domain, showedstrong reporter activity (FIG. 6), although the ΔNp63γ gave a low butdetectable signal. Surprisingly, however, the ΔNp63α isotype also failedto yield a significant level of reporter gene expression, despite havingthe same transactivation domain as TAp63γ. A similar lack oftransactivation was seen with TA*p63α, pointing to additional regulatoryfacets of p63 involving its C-terminal domain.

Example 12 Induction of Apoptosis by p63 Isotypes

Expression of wildtype p53 induces apoptosis in a wide variety of cells,whereas many p53 mutants have lost this ability (Oren, 1994). Todetermine whether any of the p63 isotypes possess similar death-inducingactivities, we compared the fates of baby hamster kidney (BHK) cellstransfected with p53 or p73 vectors with those expressing p63 isotypes.BHK cells were transfected with the p53, p73, and p63 expression vectorsand fixed 16 hours later. Cells were labeled using anti-Myc and anti-HAepitope tag antibodies to detect expressed proteins and with the DNAfluorochrome Hoechst 33278. Approximately 90% of the wildtypep53-expressing cells appeared raised from the substrate and showedhighly condensed, lobated nuclei characteristic of apoptotic cells (FIG.7A). In contrast, those expressing the p53(V143A) mutant showed a verylow percentage of apoptotic cells, despite high levels of exogenousprotein expression (FIG. 7B). Cells transfected with TAp637 provedhighly vulnerable to cell death, as evidenced by nuclear morphology,despite the generally low protein levels generated from exogenousexpression in these cells. However, an overexposure of the epitope-tagimmunofluorescence image showed a good correspondence between apoptoticcells and TAp63γ expression (FIG. 7C). Curiously, cells expressing theΔNp63γ isotype, which yielded low but measurable activity in theβ-galactosidase assays (FIG. 6), also exhibited a slight but noticeablelevel of apoptosis. The percentage of cell death induced by ΔNp63γhowever, was considerably less than those seen in cells expressing p53or TAp63γ, despite the fact that ΔNp63γ accumulates to very high levelsin BHK cells (FIG. 7D). Apoptosis was virtually absent in cellsexpressing high levels of either ΔNp63α (FIG. 7E), TAp63α, or TA*p63α,consistent with the lack of transactivation seen with these variants.Finally, p73α and p73β exhibited little or no apoptotic activity inthese cells at 16 hours, despite high levels of accumulation.

Example 13 ΔN-p63γ Suppresses Transactivation by p53, and Enhances thatof TA-p63γ

The ability of the TAp63γ isotype to transactivate reporter genesbearing p53-responsive elements suggested that, in general, p63 isotypescan interact with p53 DNA binding sites. As the ΔNp63γ isotypes lack theacidic N-terminus similar to that required for transcriptionalactivation by p53, it seemed feasible that such isotypes could act in adominant-negative manner towards both p53 and transactivating versionsof p63, such as TAp63γ. To test whether ΔNp63γ isotypes could in factsuppress the transactivation ability of p53, we transfected Saos-2 cellswith a constant amount wildtype p53 and varying concentrations of eitherΔNp63γ or ΔNp63α and assayed for transactivation of the PG-13 β-galreporter gene. At a 5:1 DNA ratio of p53 and ΔNp63γ transfected intoSaos-2 cells, reporter activity was reduced to 37% that of p53 alone,while a 1:1 ratio yielded less than 20% the transactivation of p53 (FIG.8A). ΔN-p63a also showed a similar, dose-dependent inhibition of p53transactivation, with the higher suppressor concentration (1:1) givingnear background (vector alone) levels of reporter signal (FIG. 8A).

We next asked if ΔN-p63 isotypes could likewise affect transactivationby TAp63γ. Paradoxically, cells co-transfected with TAp63γ and ΔNp63γ(5:1) yielded reporter expression slightly above that seen with TAp63γalone (FIG. 8B). Higher levels of ΔNp63γ in the cotransfection (1:1ratio) suppressed transactivation by TA-p63γ by a modest 20% (FIG. 8B).In contrast, ΔN-p63α proved to be a strong suppressor of transactivationby TAp63γ, yielding only background levels of reporter signal, even whenco-transfected at one-fifth the DNA concentration of TAp63γ (FIG. 8B).

Several mechanisms could underlie the ability of ΔNp63 isotypes tosuppress p53 and p63 in these assays. For example, given the high degreeof sequence homology within the DNA binding domains of the p53 and p63proteins, it is likely that p63 can bind p53 DNA target sites in acompetitive manner. To address this possibility, we asked whether p63isotypes, particularly those lacking detectable transactivationcapabilities, could nonetheless interact with p53 DNA binding sites.Electrophoretic mobility shift assays (EMSA) were performed using threeseparate oligonucleotides: a minimal p53 binding sequence site (PG), ap53 binding site in the p21 promoter (WAF), and a mutant p53 bindingsite (MG; Kern et al., 1992) with lysates of 293 human kidney cellstransfected with p53, ΔNp63γ, TAp63α and green fluorescent protein. p53,ΔNp63γ and TAp63α lysates all yielded significant shifts of both PG andWAF oligonucleotides, while GFP, included as a negative control, failedto display a similar shift (not shown). None of the lysates showed ashift of the control, non-p53 binding oligonucleotide, MG, thusdemonstrating the specificity of p53, ΔNp63γ and TAp63α. interactionswith the p53-binding sites.

Another mechanism by which p63 isotypes could affect transactivation byp53 and p63 is via direct protein-protein interactions, presumablythrough their oligomerization domains, We tested the potential for suchinteractions using a glutathione S-transferase (GST), TA*p63γ fusionconstruct (GST-TA*p63γ). Co-expression in BHK cells and subsequentbinding assays showed strong associations between GST-TA*p63γ and p63γisotypes, including and ΔNp63α but failed to reveal an interaction withp53.

Example 14 Electrophoretic Mobility Shift Assays (EMSA)

This invention provides nucleic acids encoding a DNA binding domain of ap63 cell regulator protein. Assays for determining the location of a DNAbinding domain in proteins include gel retardation assays, well known inthe art. Briefly, recombinant proteins comprising various portions of ap63 cell regulator protein can be produced and their interaction withDNA can be measured by incubation with a DNA target sequence andseparation of the complexes by gel electrophoresis. The DNA targetsequence of a p63 cell regulator protein can be determined, e.g., bybinding site selection experiments, well known in the art. Binding siteselection experiments are performed by incubation of a DNA bindingprotein, e.g., a p63 cell regulator protein with a degenerate pool oflabeled double stranded oligonucleotides and isolation of theoligonucleotides which interact specifically with the DNA bindingprotein. Individual oligonucleotides are then sequenced.

EMSAs were performed essentially as described in Yang, A. et al.,(1998), Mol Cell 2, 305-316. Briefly, human 293 kidney cells weretransfected with p53, p63, and GFP expression vectors, as indicated inFIG. 25, using the calcium phosphate transfection method previouslydescribed (Heald et al., 1993, Cell 74, 463-474; Yang et al., 1998).Cells were lysed in 150 μl detergent lysis buffer (50 mM Tris pH 8, 150mM NaCl, 0.1% Triton X-100) ˜24 hrs after transfection. Lysates werethen incubated for 1 hr at room temperature with 100 μM ³²Pradiolabeled, double-stranded oligonucleotides in binding buffer (16 mMHepes-KOH pH 7.5, 60 mM KCl, 30 mM NaCl, 10% glycerol, 1 mMdithiothreitol, 10 μg/ml BSA). The following oligonucleotides were used,with annealing of oligonucleotide pairs performed prior to incubationwith lysate extracts above.

PG: 5′-CCTGCCTGGACTTGCCTGG + (SEQ ID No: 35) 5′-CCAGGCAAGTCCAGGCAGG (SEQID No: 36). WAF: 5′-GAACATGTCCCAACATGTTG + (SEQ ID No: 37)5′-CAACATGTTGGGACATGTTC (SEQ ID No: 38). MG: 5′-CCTTAATGGACTTTAATGG +(SEQ ID No: 39) 5′-CCATTAAAGTCCATTAAGG (SEQ ID No: 40).

Example 15 Induction of p63 in Response to UV/DNA Damage

These experiments show that like p53 p63 is induced in response tostress signals such as UV-DNA damage.

RT-PCR Analysis

Total RNA was isolated from tissues and cell lines using RNAzol,dissolved in 10 mM Tris pH8, 1 mMEDTA (TE), and quantified usingultraviolet absorption at 260 nm. RT-PCR reactions were performed withthe One-Step RT-PCR kit (Gibco-BRL), using 0.25 μg total RNA in 25 μlreactions under the following conditions: 50° C. 30 min; 94° C. 2 min;94° C. 30 sec, 52° C. 30 sec, 72° C. 1 min for 40 cycles; 72° C. 5 min.The following primers were used: human p63 TA-specific reaction:5′-ATGTCCCAGAGCCACACAG (SEQ ID No: 41) and 5′-AGCTCATGGTTGGGGCAC (SEQ IDNo: 42); human p63 ΔN-specific reaction: 5′-CAGACTCAATTTAGTGAG (SEQ IDNo: 43) and 5′-AGCTCATGGTTGGGGCAC (SEQ ID No: 44).

UV-Irradiation of Human Keratinocytes

Human foreskin primary keratinocytes were cultured in Keratinocyte-SFMmedia (Gibco-BRL) and maintained in 5% CO₂. The keratinocytes weretreated with 300 J/m² UV irradiation, and harvested at times indicatedfor total RNA using RNAzol, as described above. RNA from untreatedkeratinocytes obtained from the same culture was used as a control.

Differentiation of Human Keratinocytes

Human foreskin primary keratinocytes were cultured in Keratinocyte-SFMmedia (Gibco-BRL) and maintained in 5% CO₂. To induce differentiation,Keratinocyte-SFM media was replaced with DMEM media (Gibco-BRL)containing 10% fetal bovine serum (FBS). Cells were harvested for RNAafter addition of DMEM/10% FBS at times indicated. RNA from untreatedkeratinocytes obtained from the same culture was used as a control.

Example 16 Screening for Mutations of the p63 Gene in Human Tumors andDiseases

Direct sequencing, using standard techniques, of the p63 gene will yieldinformation on the genomic organization (i.e. intron/exon boundaries)and nucleotide sequence for exons, introns and promoter regions of p63.An important biological and diagnostic application for these data willbe to screen for sequence mutations and/or polymorphisms (includingnucleotide substitutions, insertions, or deletions) that may result in aloss or gain of function of the p63 gene. These screens will employ theuse of techniques standard in the field, including, but not restrictedto, single strand conformation polymorphism (SSCP) analysis, and directsequencing of DNA or RNA samples obtained from patients.

As one means of enabling this application, we have isolated a PAC cloneof an approximately 120 kilobase genomic segment containing the p63gene. Briefly, an 800 bp amplicon, derived from PCR on human genomic DNAand corresponding to portions of exon 7 and 8 and intervening intron,was used as a probe in hybridization screens for the human p63 gene.Screening (done by Genome Systems) of a human genomic PAC library (madefrom white blood cells, male) yielded one clone containing the p63 gene.We have confirmed the identity of this PAC clone using by PCR. Hup63geno(PAC) has been deposited at the ATCC (10801 University Blvd., Masassas,Va. 20110) under the terms of the Budapest Treaty. The deposit was madeon Oct. 13, 1997 and received ATCC accession number 209359. TheHup63geno (PAC) clone likely contains a majority of the p63 gene, as theDNA probe used hybridizes to a core, central domain of the gene.Regardless, the sequence information, as well as the use of DNA probesderived from this PAC clone render the isolation of any portion of thep63 gene missing from this clone a standard and obvious application.

Example 17 Lineage-Specific Expression of p63 in Genital Tract Neoplasia

Vulvar, cervical, endometrial, and ovarian epithelial neoplasms, mixedmullerian tumors (MMMT), stromal sarcomas and adjacent normal epitheliawere studied. Serial sections were stained with monoclonal antibodies top63 and p53. Percentages of cells staining were estimated for eachneoplastic phenotype. It was found that in the vulva or cervix, p63expression was limited to squamous epithelium and reserve cellpopulations. Staining was uniformly negative in benign and neoplasticendocervical glandular epithelium. Staining was weak (less than 10%) orabsent in all but 2 endometrial adenocarcinomas, in all MMMTs, and inall ovarian neoplasms except one low grade transitional carcinoma. Whenpresent in adenocarcinomas, p63 staining predominated in basal/reservetype cells and foci of squamous metaplasia. P53 expression wasconspicuous (>10%) only in endometrial serous carcinomas (12/16), onestromal sarcoma, and one MMMT, and did not co-localize with p63.

Therefore, it appears that p63 is a unique homologue of p53 which, inthe cervix, is expressed exclusively in squamous epithelium or reservecells. In glandular lesions of other sites, p63 predominates in reservecells, areas of squamous differentiation and basal cell populations. Thesharp differences in expression of p63 between glandular and squamousepithelium, particularly in the cervix, may provide insights into themechanisms determining phenotype in both benign and neoplasticepithelial proliferations.

Example 18 p63 is a Differentiation Specific Marker in Cervical SquamousEpithelium

The distribution of p63 expression in a range of cervical squamousepithelia was examined and contrasted it with markers for cellproliferation (Ki-67) because p63 is homologous to p53 and p53regulation has been implicated in the pathogenesis of HPV-relatedsquamous neoplasia.

31 biopsies classified as reactive, atrophic, intraepithelial andinvasive cervical squamous epithelial alterations, as well as normalmucosa, were analyzed by immunohistochemistry for p53 and Ki-67 fordistribution and correlation with morphologic phenotype. It was foundthat distribution of p63 closely paralleled squamous celldifferentiation, staining all nuclei in the lower one third to one halfof normal squamous epithelium only. Diffuse staining of all epithelialcells occurred in immature epithelia, including atrophy, immaturemetaplasia, immature LSILs (papillary immature metaplasia) and theimmature cells of conventional low and high grade SIL and invasivecancer. In contrast, Ki-67 staining was more diffuse in neoplasticlesions, being expressed in both differentiated and undifferentiatedcell nuclei, and less frequently expressed in benign processes.

Therefore, it appears that p63 is a unique homologue of p53 which, inthe cervix, is expressed almost exclusively in immature squamousepithelium irrespective of the pathologic process. The morphological andimmunohistochemical evidence are consistent with a role of p63 in celldifferentiation, uncoupled from both cell proliferation and HPVexpression. Cessation or down-regulation of p63 expression may play acritical role in the process of squamous differentiation, both benignand neoplastic.

1. A nucleic acid sequence encoding a p63 cell regulatory protein,wherein said: nucleic acid hybridizes under stringent conditions to anucleic acid of SEQ ID Nos: 1-12, wherein said p63 cell regulatoryprotein binds a target DNA sequence.